Abstract

RASSF1A is a putative tumor suppressor whose role in cell lines of epithelial origin has been extensively studied. Though RASSF1A has been shown to be epigenetically silenced in a variety of leukemia and lymphoma tumor lines, its role in lymphocyte proliferation and apoptosis has been the subject of considerably less investigation. Interaction of Fas ligand (FasL) on activated T lymphocytes with the Fas receptor on other T cells or other immune cells plays a major apoptotic role in controlling an immune response through activation-induced cell death (AICD). Hence, our research efforts have focused on examining the effect of RASSF1A on FasL-mediated apoptosis in the Jurkat human T cell line. Our preliminary co-transfection studies demonstrated that over-expression of RASSF1A induced a 2.5-fold increase in FasL promoter (FasLp) activity. Similar-fold increases in activity were observed in the distal NFAT binding site in FasLp as well as the NFAT/AP1 site in IL-2p, indicating that RASSF1A’s effect on NFAT is not unique to FasLp sites. RASSF1A does not appear to affect the NFAT regulator, calcineurin, suggesting that it acts through an alternative NFAT activation pathway. Experiments with mutant Jurkat cell lines indicate that early signaling mediators, including tyrosine kinases and PLC-γ, are critical in the RASSF1A-induced enhancement of FasL production. Additional studies with FasLp response elements implicated in AICD, such as FLRE, will determine whether RASSF1A enhances FasL transcriptional activity exclusively through the distal NFAT site. We have created a Jurkat cell line that stably expresses high levels of RASSF1A, and a control cell line stably transfected with vector-only. Ongoing studies will focus on differences in cytotoxicity, or ability to induce fratricide in wild-type target Jurkat cells, between the vector control and the RASSF1A-expressing cell lines.