A genetic strategy involving a glycosyltransferase promoter and a lipid translocating enzyme to eliminate cancer cells.

The most common therapeutic strategy for the treatment of cancer uses antimetabolites, which block uncontrolled division of cancer cells and kill them. However, such antimetabolites also kill normal cells, thus yielding detrimental side effects. This emphasizes the need for an alternative therapy, which would have little or no side effects. Our approach involves designing genetic means to alter surface lipid determinants that induce phagocytosis of cancer cells. The specific target of this strategy has been the enzyme activity termed aminophospholipid translocase (APLT) or flippase that causes translocation of phosphatidylserine (PS) from the outer to the inner leaflet of the plasma membrane in viable cells. Efforts to identify the enigmatic, plasma membrane APLT of mammalian cells have led investigators to some P-type ATPases, which have often proven to be the APLT of internal membranes rather than the plasma membrane. By measuring kinetic parameters for the plasma membrane APLT activity, we have shown that the P-type ATPase Atp8a1 is the plasma membrane APLT of the tumorigenic N18 cells, but not the non-tumorigenic HN2 (hippocampal neuron x N18) cells. Targeted knockdown of this enzyme causes PS externalization in the N18 cells, which would trigger phagocytic removal of these cells. But how would we specifically express the mutants or antisense Atp8a1 in the cancer cells? This has brought us to a glycosyltransferase, GnT-V, which is highly expressed in the transformed cells. By using the GnT-V promoter to drive a luciferase reporter gene we have demonstrated a dramatic increase in luciferase expression selectively in tumor cells. The described strategy could be tested for the removal of cancer cells without the use of antimetabolites that often kill normal cells.

Isolation, sequencing, and functional analysis of the TATA-less murine ATPase II promoter and structural analysis of the ATPase II gene.

The P-type Mg2+-ATPase, termed ATPase II (Atp8a1), is a putative aminophospholipid transporting enzyme, which helps to maintain phospholipid asymmetry in cell membranes. In this project we have elucidated the organization of the mouse ATPase II gene and identified its promoter. Located within chromosome 5, this gene spans about 224 kb and consists of 38 exons, three of which are alternatively spliced (exons 7, 8 and 16), giving rise to two transcript variants. Translation of these transcripts results in two ATPase II isoforms (1 and 2) composed of 1164 and 1149 amino acids, respectively. Using RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) we identified multiple transcription start sites (TSS) in messages obtained from heart, lung, liver, and spleen. The mouse ATPase II promoter is TATA-less and lacks a consensus initiator sequence. Luciferase reporter analysis of full and core promoters revealed strong activity and little cell type specificity, possibly because more flanking, regulatory sequences are required to cause such tissue specificity. In the neuronal HN2, N18, SN48 cells and the NIH3T3 fibroblast cells, but not in the B16F10 melanoma cells, the core promoter (-318/+193 with respect to the most common TSS) displayed significantly higher activity than the full promoter (-1026/+193). Serial 5' deletion of the core promoter revealed significant cell type-specific activity of the fragments, suggesting differential expression and use of transcription factors in the five cell lines tested. Additionally distribution of the TSS was organ specific. Such observations suggest tissue-specific differences in transcription initiation complex assembly and regulation of ATPase II gene expression. Information presented here form the groundwork for further studies on the expression of this gene in apoptotic cells.

Isolation, sequencing, and functional analysis of the TATA-less human ATPase II promoter.

Multiple lines of evidence indicate that the P-type Mg(2+)-ATPase, termed ATPase II, could play an important role in apoptosis. With the long-term objective of studying the regulation of this protein during apoptosis, we delineated the exon-intron organization of the human ATPase II gene (within chromosome 4). Subsequently, we used RNA ligase-mediated rapid amplification of cDNA ends to identify a major transcription start site at position -143 with respect to the translation start site. Luciferase reporter analysis of a 1.2-kb 5'-flanking sequence (-1222 to +94 with respect to the transcription start site) revealed strong promoter activity in three human cell lines, human oligodendroglioma (HOG), SHSY5Y (hybrid neuroblastoma), and EA.hy926 (endothelial cell line). Serial deletions from the 5' end of this sequence up to nucleotide -291 yielded some decrease in activity only in the EA.hy926 cells. Further deletion to -217 caused a drastic decrease in activity in all three cell lines, but a -148 fragment showed preferential reduction in activity in the EA.hy926 cells. The promoter activity was nearly equal in two sequence variants of the promoter, one of which (designated as Variant 2) contained a 15-bp direct repeat within a GC-rich region. Additionally, there were several single base-pair changes from the sequence reported by the human genome project. Despite the presence of enhancer/repressor elements, such as Sp1 and NFkappaB, relatively small differences in promoter activity were observed in the three cell lines. However, it is likely that such sequence elements could cause major regulation of promoter activity in cells subjected to conditions that trigger apoptosis. The ATPase II promoter sequence will provide valuable clues to the regulation and role of the ATPase II protein.

Appearance of voltage-gated calcium channels following overexpression of ATPase II cDNA in neuronal HN2 cells.

ATPase II (a Mg2+-ATPase) is also believed to harbor aminophospholipid translocase (APTL) activity, which is responsible for the translocation of phosphatidylserine (PS) from the outer leaflet of the plasma membrane to the inner. To test this hypothesis we overexpressed the mouse ATPase II cDNA in the neuronal HN2 cells. In addition to a dramatic increase in APTL activity, we also made the unexpected observation that expression of the mouse ATPase II cDNA from the vector pCMV6 resulted in the appearance of calcium current. Although the hybrid cell line HN2 or a line (HN2V32) obtained by expressing a heterologous gene from the same expression vector showed no calcium current, both ATPase II-overexpressing clones (HN2A12 and HN2A22) showed significant barium conductance. This current was due to calcium channels because it was blocked almost completely by 100 microM CdCl2 and it had a significant N-type component since it was blocked by 38.5% in the presence of 5 microM omega-conotoxin (omega-CTX). Western blot analysis using an antibody against the N-type calcium-channel alpha1B subunit revealed a dramatic increase in expression of this protein in the HN2A12 and HN2A22 cell lines. Our results suggest that ATPase II also harbors APTL activity. In view of the prior knowledge that APTL activity is inhibited by an increase in calcium, our results also suggest that APTL expression exerts a negative feedback regulation on itself by inducing expression of channels that cause an influx of calcium ions. The mechanism of this regulation could reveal important information on a possible cross-regulation between these two families of proteins in neuronal cells.