The overall goal of this five year research plan is to test the Hypothesis: TCDD acts directly on B-cells to suppress immunoglobulin secretion. This suppression is mediated by the AhR which adversely regulates immunologically relevant genes possessing DREs in their 5' regulatory regions. Toward characterizing the putative role by the AhR in TCDD-mediated B-cell dysfunction, our results have shown that mouse leukocytes and purified B-cells express functional Ah receptor and ARNT as evidenced by (a) Western blot; (b) increased DRE binding in the presence of TCDD, and (c) induction of cytochrome-P450 1A1 (Cyp1A1) gene expression. Moreover, we demonstrated that activation of mouse splenocytis, or primary B-cells, produced a marked increase in AhR mRNA and protein following activation. The significance of AhR up-regulation is unclear but may explain the sensitivity of immunocompetent cells to TCDD. Identical results were observed in the CH12.LX B-cell line following LPS activation. In the presence of TCDD CH12.LX cells exhibited inhibition in LPS-induced immunoglobulin secretion and induction of Cyp1A1. A second B-cell line, BCL-1, possesses ARNT but is AhR-deficient. BCL-1 cells in the presence of TCDD exhibited no inhibition of LPS-induced immunoglobulin secretion and no induction of Cyp1A1. Most recently, we have also demonstrated that TCDD induced a marked increase in the tumor suppressor gene and transcription factor, p53 which we believe plays a critical role in B-cell dysfunction by TCDD. In light of the above findings. We will test our hypothesis using the following specific aims (SA): In SA number1, we will characterize the structure activity relationship between TCDD-mediated inhibition of immunoglobulin secretion and heavy chain mRNA expression versus the regulation of gene transcription through the DRE. In SA number2, we will determine whether transfection of the AhR into an AhR-deficient cell-line (BCL-1) can confer sensitivity to TCDD. In SA number3, we will determine whether TCDD treatment of B-cells interferes with the ability of the transcription factor, B-cell specific activator protein (BSAP), in binding to its consensus recognition sequence. In, SA number4, we will characterize the role of premature p53 upregulation by TCDD in B-cells on cell cycle progression and immunoglobulin secretion. Lastly, in SA number5 we will determine whether up-regulation of p53 by TCDD in B-cells is mediated through the binding of the AhR/ARNT complex to a DRE-like site in the p53 promotor.
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