The immortalization of B lymphocytes may represent a pre-neoplastic stage of malignant transformation. It occurs by unknown mechanisms which may involve altered function of growth-regulatory cellular genes, eg. oncogenes. The objective of this proposal is to study the mechanism of in vitro immortalization of human B lymphocytes by Epstein-Barr virus (EBV) in order to better understand the genesis of B cell malignancies. During phase I (2 years) cellular and molecular alterations that occur with B cell immortalization will be examined in detail by comparing nontransformed clonal B cell lines with isogenic immortalized lines. Clonal lines of nontransformed, normal B cells which are B cell growth factor (BCGF)-dependent will be established by limiting dilution and soft agar cloning methods. Isogenic immortalized lines will be derived from the normal B cell lines by in vitro EBV infection. Comparisons of growth properties, chromosomal abnormalities, and oncogene expression will be made between normal B cells and isogenic EBV-transformed B cells. B cell lines which spontaneously transform to BCGF-independent proliferation in the absence of EBV will be similarly examined. The transcriptional expression of oncogenes may vary with the activation state of normal B cells. Therefore, oncogene expression will also be carefully examined in normal B cells during different states of activation, including resting, mitogen-stimulated, and BCGF-dependent proliferating states. Three complementary methods (""""""""in situ"""""""" hybridization, RNA """"""""dot blots,"""""""" and northern blots) will be used to assess expression of several oncogenes, incuding myc and B-lym. Phase II studies will focus on the molecular mechanism of EBV immortalization by identifying the region(s) of the EBV genome required for immortalization. EBV genomic fragments cloned in Charon 4A phage will be introduced into normal BCGF-dependent B cells by electroporation. Fragments which encode growth transforming functions can be identified by outgrowth of BCGF-independent B cells. The transforming region(s) can be further delimited by identifying subfragments which retain transforming functions. Functional mapping of the EBV transforming region(s) by this approach may ultimately lead to the identification of specific transforming genes and their products.
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