Chemotherapy causes apoptosis of brain tumor cells, which are then cleared through phagocytosis. In peripheral tissue, phagocytosis is preceded by a recognition process in which scavenger macrophages selectively bind to phosphatidylserine (PS) molecules that are exposed on the surface of the apoptotic cells. During the current granting period, the applicant's team has shown that PS plays a central role also in the recognition of apoptotic neurotumor cells by the resident scavenger cells of the brain, the microglia. The enzyme that plays a central role in the inner-membrane sequestering of PS in healthy cells is the aminophospholipid translocase (APTL), which is also a Mg2+-ATPase and belongs to a recently classified subfamily of P-type ATPase. It is highly expressed in the CNS, but its regulation and role in neurons are poorly understood. Its inhibition or down regulation results in the typical apoptotic feature of PS-externalization. In order to study the regulation profile of this enzyme, the mouse APTL cDNA was overexpressed from vector pCMV6 in the calcium channel-deficient hybrid neuroblastoma cells, HN2. In addition to showing a 15-fold increase in phosphatidylserine translocase activity, all the clones surprisingly displayed significant levels of voltage-gated calcium channels. Another cell line (HN2V32) that was prepared by stable expression of a heterologous gene also harbored in pCMV6, did not display any voltage-gated calcium current. The central goal of this renewal application is to characterize the proximal promoter of the APTL gene, and also understand the correlation between overexpressed APTL and the appearance of voltage-gated calcium channels. The proximal promoter sequence will be obtained using """"""""Rapid Amplification of cDNA Ends"""""""" (5'-RACE) and S1 nuclease digestion analysis. Functional activity and sequence features of the promoter will be tested using serially deleted segments of the promoter sequence to drive expression of the chloramphenical acetyl transferase (CAT) reporter gene. Software analysis of the promoter sequence will reveal the presence of enhancer/repressor elements. Possible synergism between APTL and pCMV6 in the expression of the pore-forming alpha1 subunit of voltage-gated calcium channels will be tested by expressing APTL cDNA from a vector completely different from pCMV6 and then testing the APTL-overexpressing clones for the expression of alpha1 subunits. Possible cross talk between APTL and the alpha1 subunits will be tested by expressing epitope-tagged APTL and then testing the effect of APTL expression levels on the expression of calcium channel alpha1 subunits. Also, APTL expression will be suppressed by antisense treatment to look for its effect on the expression and activity of calcium channels. Results from this project will shed new light on the regulation and role of the protein APTL in brain neurons and apoptotic neurotumor cells.
