Regulation of Primary Human Beta Cell Culture by E2A1-14

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有涉及E2A基因的染色体易位，导致致癌的E2A融合蛋白的表达。这些易位几乎是pb - all所独有的，只有极少数例外。由于我们目前的实验模型存在缺陷，E2A融合蛋白如何引起pb - all尚不清楚。在小鼠和细胞系模型中，E2A融合蛋白转化成纤维细胞、骨髓细胞和T细胞，但不转化B细胞；相反，E2A融合蛋白减少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细胞的理解大多来源于对非B细胞的研究，可能不适用于B细胞。例如，目前的研究表明，截断形式的E2A融合蛋白没有活性。然而，这种形式存在于大约3 - 4%的儿科铅- all中。为了更好地理解这种截断形式和其他E2A融合蛋白，我们开发了一种新的实验模型，在该模型中，慢病毒在正常儿童前体B细胞（E2A融合蛋白转化的天然靶细胞）的原代培养物中表达截断形式。截断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的限制。我们建议使用这个实验系统：1。表征并比较实验B细胞与表达截断形式的pb - all。我们将通过免疫球蛋白基因重排和慢病毒整合位点的PCR来确定实验B细胞是单克隆还是多克隆。分化状态将通过流式细胞术进行表征，并与表达截断形式的pb - all进行比较。这些特异性的pb - all将通过RT-PCR鉴定融合转录物，并通过流式细胞术进行鉴定。2. 描述截断形式促进B细胞扩增/存活的机制。我们将使用流式细胞术和PCR ELISA测定E2A融合蛋白的截断形式是否增加细胞分裂，减少细胞凋亡，或增加端粒酶活性

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