C-MYC EXPRESSION AND APOPTOSIS IN A B CELL LINE

B 细胞系中的 C-MYC 表达和凋亡

• 批准号: 2089092
• 负责人: GAIL E. SONENSHEIN
• 金额: $ 19.96万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-01-01 至 1997-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2089092
• 关键词: B lymphocyte; DNA binding protein; antiantibody; apoptosis; gene expression; gene mutation; genetic promoter element; genetic transcription; immune tolerance /unresponsiveness; molecular cloning; phosphorylation; protein sequence; protooncogene; tissue /cell culture

Apoptosis or programmed cell death (PCD) is a general mechanism of cell suicide that has been implicated in tolerization of B lymphocytes. For example, treatment of the murine early B lymphoma WEHI 231 line, a model for study of B cell tolerance, with an antiserum against its expressed surface IgM, such as goat anti-mouse Ig (GaMIg) or anti-mu antisera, inhibits its proliferation via apoptosis. Oligosomal degradation of DNA is detectable by 12 hours post-GaMIg treatment. Recent evidence has demonstrated that the expression of the nuclear proto-oncogene c-myc is necessary for PCD, promoting apoptosis in a dose-dependent fashion. Here the role of c-myc in apoptosis will be explored using the WEHI 231 line as model system. The expression of c-myc in WEHI 231 cells and the effects of apoptosis have been extensively characterized by ongoing work from the PI's and other laboratories. Treatment of WEHI 231 cells with GaMIg results in an initial increase in expression of c-myc RNA of 5- to 10-fold by 1-2 hours, which is followed by a dramatic decline by 4-6 hours post-treatment. The synthesis of c-myc protein parallels the early changes in RNA levels; furthermore, the protein is transiently hyperphosphorylated at 1 hour. By 24 hours, mRNA and protein levels are well below those observed in control cells. A major site of control of these changes in c-myc RNA expression is mediated at the transcription level. The increase in c-myc expression appears to be critical for apoptosis. For example, the PI's laboratory has recently shown that treatment with an anti-delta antiserum of a WEHI 231 line stably transfected with a delta heavy chain (WEHI 231-delta), which fails to induce apoptosis, failed to induce c-myc protein levels, in contrast to treatment with anti-mu serum. Thus the aims of this proposal are to 1) measure the effects of anti-Ig treatment on c-myc protein expression, and 2) characterize the transcription factors mediating the changes in c-myc RNA levels; particular emphasis, will be placed on the nuclear factor NF-KB, which the PI's laboratory has demonstrated plays a major role in regulation of c-myc transcription. Specifically, post-translational modifications and association of c-myc with other cellular proteins will be examined. The biochemical nature and functional effects of changes in expression of NF-KB, noted during apoptosis, will be measured. Results will be correlated with apoptosis through use of the WEHI 231-delta line. These studies should provide important insights into the control of apoptosis in lymphocytes, the development of B cell tolerance and the role of the c-myc oncogene in these processes.

细胞凋亡或程序性细胞死亡（PCD）是细胞凋亡的一般机制。 与B淋巴细胞耐受有关的自杀。 为 例如，治疗小鼠早期B淋巴瘤WEHI 231系，一种模型 为研究B细胞耐受性，用抗其表达的抗血清， 表面IgM，例如山羊抗小鼠IG（加米格）或抗μ抗血清， 通过凋亡抑制其增殖。 DNA的寡聚体降解 在GaMIg处理后12小时可检测到。 最近的证据 证明了核原癌基因c-myc的表达是 这是PCD所必需的，以剂量依赖性方式促进凋亡。 这里 利用WEHI 231细胞系探讨c-myc在细胞凋亡中的作用 作为模型系统。 c-myc在WEHI 231细胞中的表达及其与细胞凋亡的关系 细胞凋亡的影响已经被正在进行的工作广泛地表征， 从私家侦探和其他实验室拿来的 用以下物质处理WEHI 231细胞： 加米格导致c-myc RNA表达的初始增加5- 10倍。 10-1-2小时，随后急剧下降4-6倍 治疗后3小时。 c-myc蛋白的合成与早期的 RNA水平的变化;此外，蛋白质是瞬时的 在1小时时过度磷酸化。 到24小时，mRNA和蛋白质水平 远低于在对照细胞中观察到的那些。 一个主要的控制点， c-myc RNA表达的这些变化是在转录时介导的， 水平 c-myc表达的增加似乎是至关重要的， 凋亡 例如，PI的实验室最近表明， 用WEHI 231系的抗δ抗血清稳定处理 用δ重链（WEHI 231-δ）转染，其不能 诱导细胞凋亡，不能诱导c-myc蛋白水平，与 抗mu血清治疗。 因此，本提案的目的是：1） 测量抗Ig处理对c-myc蛋白表达的影响，和 2)鉴定介导c-myc变化的转录因子 RNA水平;特别强调，将放在核因子 NF-κ B，PI的实验室已经证明了在 c-myc转录的调控。 特别是翻译后 c-myc与其它细胞蛋白的修饰和结合将 接受检查。 变化的生物化学性质和功能效应 将测量在细胞凋亡期间记录的NF-κ B的表达。 结果 将通过使用WEHI 231-δ系与细胞凋亡相关。 这些研究应该为控制 淋巴细胞凋亡、B细胞耐受性的发展以及 c-myc癌基因在这些过程中的作用。