In the natural process known as phagocytosis a multicellular organism removes unwanted or injured cells with the help of scavenger cells, termed phagocytes. Such phagocytic cells, e.g. the macrophages in the peripheral system and the microglia in the brain, specifically bind to the dying cells through certain cell-surface molecules, one of which is phosphatidylserine (PS). PS, which normally resides in the inner-leaflet of the plasma membrane, flip flops to the outer leaflet in the dying cells and is recognized by PS receptors located on phagocytic cells. In healthy cells a bifunctional enzyme, which is both a Mg2+-/-ATPase and also an aminophospholipid translocase (APTL), translocates PS to the inner leaflet of the plasma membrane. This process apparently overrides the effect of two other enzymes, the scramblase (which bidirectionally translocates all phospholipids molecules) and the floppase (which very slowly translocates phospholipids from the inner leaflet to the outer). Many earlier studies have shown that offsetting the balance among APTL, scramblase, and floppase by inhibiting APTL results in externalization of PS and phagocytosis. A P-type ATPase, ATPase II, bears striking similarity in properties to APTL and transfection of antisense ATPase II cDNA causes externalization of PS. Expression of ATPase II mRNA shows tissue-specificity, with the highest levels of expression observed in the brain and the skeletal muscle. We have isolated the ATPase II promoter and showed that it displays considerable tissue specificity in luciferase reporter assays. In our current project we will further analyze the promoter of the ATPase II gene to study the sequence elements that confer this cell type-specificity of expression. Also, in our earlier studies, we had observed that stable overexpression of ATPase II causes appearance of mixed, voltage-gated calcium channels in a hippocampal neuron-derived cell line, HN2, which normally does not display any calcium current. In parallel, induced expression of the channel-forming subunit, (1B, of the N-type channels was also observed. We will prepare and test an inducible expression system for ATPase II in the HN2 cells in order to verify if this induced expression of calcium channels was due to transactivated gene expression or a protein-protein interaction between ATPase II and the calcium channel proteins. Finally, since antisense ATPase II transfection causes externalization of PS molecules, we will use this concept to design and test a novel strategy of removing cancer cells through phagocytosis without attempting to kill them with antimetabolites. In this strategy we will selectively ablate ATPase II expression in the cancer cells by using ATPase II-specific small inhibitory RNA (siRNA). Selective expression of the siRNA in cancer cells will be achieved through the use of a promoter for a highly cancer cell-selective protein, e.g. the (1-6 N acetyl glucosaminyl transferase V (GnT-V). Abrogation of ATPase II in the cancer cells would cause PS externalization and phagocytic removal of these cells. Studying the mechanism of ATPase II expression, analyzing its role in the induction of calcium channels, and evaluating its use in designing a new strategy for the removal of cancer cells will be the overall goal of our project.
