The goal of this project is to study the mechanisms of viral oncogene (v-onc) induced growth deregulation in vitro, using as model systems mature murine lymphocytes whose activation and growth characteristics are well-defined. Oncogenes will be introduced into antigen-specific T cell lines and splenic B cells by infection with acute transforming retroviruses pseudotyped with an amphotropic murine leukemia virus. Preliminary experiments using Ki-ras carrying viruses have shown that by this method, both B and T lymphocyte lines can be generated that express v-onc and exhibit alterations in activation and growth response compared to their uninfected counterparts. The proposed project uses the following approaches to examine issues that are relevant to growth deregulation in all cells that require exogenous stimuli or factors for sustained growth. 1. The technique of viral infection of mature lymphocyte populations in vitro will be refined using pseudotyped acute transforming retroviruses carrying Ki ras and other v-onc, in order to establish panels of T and B lymphocyte lines with different phenotypic and functional properties. 2. Phenotypic markers on these lines will be examined in order to assess the effects of v-onc on surface molecules that are related to cellular maturation, activation, growth and effector functions. 3. The virus-infected lines will be assayed for dependence on and responsiveness to defined growth factors, e.g. interleukin 1 and interleukin 2 for T cells, and comparisons with uninfected cell will be made. 4. The responses of cell lines to known activation signals, and the effects of these stimuli on intracellular """"""""messengers"""""""" (e.g. Ca++, inositol phosphates) and subsequent growth and differentiative responses will be analyzed. 5. The role of v-onc expression in producing changes in activation and growth physiology will be studied by: a) isolating subclones with different levels of v-onc expression or reversion to normal growth phenotypes and correlating these, and b) selectively inhibiting v-onc gene expression.
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