The long-term objective of this research is to define the role of the fos protein in T cell proliferation mediated through the antigen and Interleukin 2 (IL2) receptors. Murine T cells transformed with the FBJ v-fos oncogene will be used to investigate the mechanism of v-fos-mediated T cell oncogenesis, and to explore the potential role of c-fos in normal T cell growth. v-fos- transformed T cells are tumorigenic in vivo and grow indefinitely in vitro in the absence of IL2, apparently because they have acquired a novel proliferative response to fetal calf serum. They have lost the ability to proliferate in response to antigen but retain the proliferative response to IL2.
The specific aims of this proposal are: (1) To examine transcription of the c-myc, c-fos, IL2 and IL2R alpha activation genes in v-fos-transformed T cells in response to serum and to IL2; the acquisition by these cells of a novel response to serum may be central to the oncogenic mechanism. (2) To define the 5' IL2R alpha gene in v-fos-transformed T cells, which express unusually high levels of IL2R alpha mRNA. Plasmids containing successive deletions of the 5' IL2R alpha sequences linked to a reporter (CAT) gene will be introduced into normal and transformed T cells by electroporation. Transient expression of CAT activity transformed cells will be compared with transient expression of CAT activity in normal cells stimulated with T cell receptor ligands and with IL2. (3) To test whether v-fos or a protein induced by v-fos can acutely regulate transcription of the IL2R alpha gene in normal T cells, by cotransfection of an expression plasmid containing the v-fos gene with CAT reporter plasmids containing 5' IL2R alpha sequences. (4) To localize the level of inhibition of the T cell receptor response in v-fos transformed cells, by testing early and late T cell receptor functions (phosphoinositide turnover, calcium mobilization, tyrosine phosphorylation, protein kinase C translocation, and transcription of activation genes in response to T cell receptor ligands). (5) To test the potential functions of c-fos in T cell growth and transformation. The c-fos gene will be introduced into T cells under control of constitutive and inducible promoters. c-fos mRNA and protein expression levels will be related to (i) morphology, (ii) serum, antigen, and IL2 responsiveness, (iii) ability to grow in the absence of IL2, and (iv) transcription of selected activation genes in response to serum, antigen/Con A, and IL2.
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