Regulation of Primary Human Beta Cell Culture by E2A1-14

E2A1-14 对原代人 β 细胞培养的调节

• 批准号: 6959798
• 负责人: JOHN Kim CHOI
• 金额: $ 14.28万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-22 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6959798
• 关键词: B lymphocyte; acute lymphocytic leukemia; apoptosis; cell differentiation; cell proliferation; chimeric proteins; chromosome translocation; clinical research; flow cytometry; human tissue; neoplasm /cancer genetics; oncoproteins; polymerase chain reaction; tissue /cell culture

DESCRIPTION (provided by applicant): The most common cancer of childhood is acute leukemia of precursor B cells (pB-ALLs). Approximately 5- 11% of pediatric pB-ALLs have chromosomal translocations involving the E2A gene, leading to the expression of oncogenic E2A fusion proteins. These translocations are almost exclusive to pB-ALLs with only rare exceptions. How E2A fusion proteins cause pB-ALLs is not known because of the deficiencies in our current experimental models. In mouse and cell line models, E2A fusion proteins transform fibroblasts, myeloid cells, and T cells but not B cells; instead E2A fusion proteins decrease B cells numbers and cause apoptosis, a finding inconsistent with oncogenesis. Hence, much of our understanding is derived from studies of non-B cells and may not be applicable to B cells. For example, current studies indicate that the truncated form of the E2A fusion proteins has no activity. However, this form is present in approximately 3 - 4% of pediatric pB-ALLs. To better understand this truncated form and eventually other E2A fusion proteins, we developed a new experimental model in which lentiviruses are used to express the truncated form in primary cultures of normal pediatric precursor B cells, the natural target cells that are transformed by E2A fusion proteins. Expression of the truncated form resulted in the expansion/survival of the precursor B cells beyond their normal limit of culture duration. We propose to use this experimental system to: 1. Characterize and compare the experimental B cells with pB-ALLs that express the truncated form. We will determine whether the experimental B cells are monoclonal or polyclonal by PCR for the immunoglobulin gene rearrangement and for the lentiviral integration site. The differentiation state will be characterized by flow cytometry and compared to those of pB-ALLs that express the truncated form. These specific pB-ALLs will be identified by RT-PCR for the fusion transcript and characterized by flow cytometry. 2. Characterize the mechanisms by which the truncated form promote the expansion / survival of B cells. We will determine whether the truncated form of E2A fusion proteins increases cell division, decreases apoptosis, or increases telomerase activity using flow cytometry and PCR ELISA assay

描述(申请人提供)：儿童最常见的癌症是急性前体B细胞白血病(PB-ALL)。大约5%-11%的儿童PB-ALL的染色体易位涉及E2A基因，导致癌基因E2A融合蛋白的表达。这些易位几乎是PB-ALL所特有的，只有极少数例外。由于我们目前的实验模型存在缺陷，E2A融合蛋白如何导致PB-ALL尚不清楚。在小鼠和细胞系模型中，E2A融合蛋白可以转化成纤维细胞、髓系细胞和T细胞，但不能转化B细胞；相反，E2A融合蛋白会减少B细胞数量并导致细胞凋亡，这一发现与肿瘤发生不一致。因此，我们对非B细胞的许多了解都来自于对非B细胞的研究，可能并不适用于B细胞。例如，目前的研究表明，截短形式的E2A融合蛋白没有活性。然而，这种形式存在于大约3%-4%的儿童PB-ALL中。为了更好地理解这种截短形式以及最终其他E2A融合蛋白，我们开发了一个新的实验模型，在该模型中，慢病毒被用来在正常的儿科前体B细胞的原代培养中表达截断形式，正常的儿科前体B细胞是由E2A融合蛋白转化的自然靶细胞。截短形式的表达导致前体B细胞的扩增/存活超过其正常培养时间限制。我们建议使用这个实验系统：1.对实验中的B细胞和表达截断形式的PB-ALL进行表征和比较。我们将通过免疫球蛋白基因重排和慢病毒整合位点的聚合酶链式反应来确定实验中的B细胞是单克隆性还是多克隆性。分化状态将通过流式细胞仪进行表征，并与表达截短形式的PB-ALL进行比较。这些特异的PB-ALL将通过RT-PCR融合转录本进行鉴定，并通过流式细胞仪进行鉴定。2.鉴定截短型促进B细胞扩增/存活的机制。我们将使用流式细胞仪和聚合酶链式反应-酶联免疫吸附试验来确定截短形式的E2a融合蛋白是否增加了细胞分裂、减少了细胞凋亡或增加了端粒酶活性