This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goal of this proposed research project is to elucidate the role(s) of complex sphingolipids in the signal transduction pathway of 3-methylcholanthrene (MC) induction of cytochrome P450 1A1 (CYP1A1). This is important to cancer research because of the key role CYP1A1 plays in the conversion of many large, organic environmental pollutants to carcinogens in vivo. We have recently discovered that inhibition of the synthesis of complex sphingolipids by ISP1 significantly suppresses the ability of MC to induce CYP1A1. In addition, we discovered that a short-chain analog of ceramide (N-acetyl-sphingosine or C2-ceramide) not only reverses this inhibition, but also significantly enhances CYP1A1 induction by MC at the levels of enzyme activity and protein and mRNA quanitites. Therefore, the specific objectives of the next phase of this project are: (1) to determine whether complex sphingolipid modulation of mRNA levels is the result of either increased mRNA synthesis or enhanced mRNA stability; (2) to determine whether sphingolipids effect the ability of other polycyclic aromatic hydrocarbons to induce CYP1A1; (3) to determine changes (if any) in the amount of AHR and ARNT after cells are treated with MC, MC+ISP1, MC+ISP1+C2-ceramide; (4) to determine other pathway interactions for sphingolipids, such as involvement in AHR-MC association, AHr-Hsp 90 dissociation, AHR-MC passage across the nuclear membrane, AHR-MC-ARNT association in the nucleus or AHR-MC-ARNT binding to an XRE on the CYP1A1 gene; (5) to determine the structural specificity of the effects of ceramide on the expression of CYP1A1, both with respect to the nature of the ceramide (e.g., the types of long-chain base backbones, the length of the amide-linked fatty acid and other types of compounds, including structural analogs); and (6) to determine whether complex sphingolipids modulate the expression of other isoforms of P450 and enzymes linked to the AHR-dependent signal transduction pathway. These studies will provide fundamental information about a potentially important pathway for the regulation of CYP1A1 and other xenobiotic metabolizing enzymes. These studies may also provide new insights into how sphingolipids affect cell behavior, particularly at the level of gene expression, and may determine how sphingolipids might be critical to cancer therapeutics.
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