Apoptosis or programmed cell death (PCD) is a general mechanism of cell suicide that has been implicated in tolerization of B lymphocytes. For example, treatment of the murine early B lymphoma WEHI 231 line, a model for study of B cell tolerance, with an antiserum against its expressed surface IgM, such as goat anti-mouse Ig (GaMIg) or anti-mu antisera, inhibits its proliferation via apoptosis. Oligosomal degradation of DNA is detectable by 12 hours post-GaMIg treatment. Recent evidence has demonstrated that the expression of the nuclear proto-oncogene c-myc is necessary for PCD, promoting apoptosis in a dose-dependent fashion. Here the role of c-myc in apoptosis will be explored using the WEHI 231 line as model system. The expression of c-myc in WEHI 231 cells and the effects of apoptosis have been extensively characterized by ongoing work from the PI's and other laboratories. Treatment of WEHI 231 cells with GaMIg results in an initial increase in expression of c-myc RNA of 5- to 10-fold by 1-2 hours, which is followed by a dramatic decline by 4-6 hours post-treatment. The synthesis of c-myc protein parallels the early changes in RNA levels; furthermore, the protein is transiently hyperphosphorylated at 1 hour. By 24 hours, mRNA and protein levels are well below those observed in control cells. A major site of control of these changes in c-myc RNA expression is mediated at the transcription level. The increase in c-myc expression appears to be critical for apoptosis. For example, the PI's laboratory has recently shown that treatment with an anti-delta antiserum of a WEHI 231 line stably transfected with a delta heavy chain (WEHI 231-delta), which fails to induce apoptosis, failed to induce c-myc protein levels, in contrast to treatment with anti-mu serum. Thus the aims of this proposal are to 1) measure the effects of anti-Ig treatment on c-myc protein expression, and 2) characterize the transcription factors mediating the changes in c-myc RNA levels; particular emphasis, will be placed on the nuclear factor NF-KB, which the PI's laboratory has demonstrated plays a major role in regulation of c-myc transcription. Specifically, post-translational modifications and association of c-myc with other cellular proteins will be examined. The biochemical nature and functional effects of changes in expression of NF-KB, noted during apoptosis, will be measured. Results will be correlated with apoptosis through use of the WEHI 231-delta line. These studies should provide important insights into the control of apoptosis in lymphocytes, the development of B cell tolerance and the role of the c-myc oncogene in these processes.
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