Regulation of Primary Human Beta Cell Culture by E2A1-14

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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