Transformation in retroviral systems is characterized by abnormal cell growth and differentiation. Expression of the v-abl protein tyrosine kinase encoded by Abelson murine leukemia virus (Ab-MLV) induces transformation in pre-B lymphocytes. These transformed cells are arrested at the pre-B cell stage based upon their immunoglobulin (Ig) gene rearrangements and cell surface antigens. Cells transformed by a temperature sensitive (ts) mutant of Ab-MLV undergo a high frequency of Ig light chain gene rearrangement soon after inactivation of the v-abl kinase at nonpermissive temperature. In addition, up-regulated expression of at least three genes important to Ig gene rearrangement (RAG-1, RAG-2 and germline kappa gene) occurs at the nonpermissive temperature. This system provides an opportunity to study the pathways mediating differentiation arrest as a component of retroviral transformation. The goals of this study are: (1) To identify functional domains of v-abl protein which mediate differentiation arrest and the pathways used to transmit these signals. A series of abl mutants and protein tyrosine kinases will be used in genetic complementation studies to determine which functional domains mediate this process. To identify pathways by which differentiation arrest is mediated, over-expression of c-ras, c-raf and c-myc will be tested, as will dominant negative forms of these genes. Proteins to be tested will be co-expressed with ts v-abl in a single pre-B cell; the differentiation phenotype of these cells will then be assessed after inactivation of the ts v-abl kinase. (2) To determine what genes are up-regulated upon release from differentiation arrest. The v-abl protein appears to block light chain rearrangement by suppressing expression of genes important for this process. The increased expression of RAG-1, RAG-2, and germline kappa gene in ts transformants at nonpermissive temperature supports this idea. These events are necessary, but probably not sufficient, to initiate rearrangement. The differential display technique will be used to identify other genes with altered expression prior to Ig rearrangement. Combining efforts to investigate both mechanisms of regulation and their targets may contribute to a better understanding of normal pre-B cell differentiation and how disruption of this process contributes to the transformed phenotype. This may provide clues to therapeutic strategies for human diseases of abnormal B lymphocyte maturation, including immunodeficiencies and both abl and non-abl related lymphoid malignancies.
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