ATP11B mediates platinum resistance in ovarian cancer.

Platinum compounds display clinical activity against a wide variety of solid tumors; however, resistance to these agents is a major limitation in cancer therapy. Reduced platinum uptake and increased platinum export are examples of resistance mechanisms that limit the extent of DNA damage. Here, we report the discovery and characterization of the role of ATP11B, a P-type ATPase membrane protein, in cisplatin resistance. We found that ATP11B expression was correlated with higher tumor grade in human ovarian cancer samples and with cisplatin resistance in human ovarian cancer cell lines. ATP11B gene silencing restored the sensitivity of ovarian cancer cell lines to cisplatin in vitro. Combined therapy of cisplatin and ATP11B-targeted siRNA significantly decreased cancer growth in mice bearing ovarian tumors derived from cisplatin-sensitive and -resistant cells. In vitro mechanistic studies on cellular platinum content and cisplatin efflux kinetics indicated that ATP11B enhances the export of cisplatin from cells. The colocalization of ATP11B with fluorescent cisplatin and with vesicular trafficking proteins, such as syntaxin-6 (STX6) and vesicular-associated membrane protein 4 (VAMP4), strongly suggests that ATP11B contributes to secretory vesicular transport of cisplatin from Golgi to plasma membrane. In conclusion, inhibition of ATP11B expression could serve as a therapeutic strategy to overcome cisplatin resistance.

Transbilayer phospholipid movements in ABCA1-deficient cells.

Tangier disease is an inherited disorder that results in a deficiency in circulating levels of HDL. Although the disease is known to be caused by mutations in the ABCA1 gene, the mechanism by which lesions in the ABCA1 ATPase effect this outcome is not known. The inability of ABCA1 knockout mice (ABCA1-/-) to load cholesterol and phospholipids onto apoA1 led to a proposal that ABCA1 mediates the transbilayer externalization of phospholipids, an activity integral not only to the formation of HDL particles but also to another, distinct process: the recognition and clearance of apoptotic cells by macrophages. Expression of phosphatidylserine (PS) on the surface of both macrophages and their apoptotic targets is required for efficient engulfment of the apoptotic cells, and it has been proposed that ABCA1 is required for transbilayer externalization of PS to the surface of both cell types. To determine whether ABCA1 is responsible for any of the catalytic activities known to control transbilayer phospholipid movements, these activities were measured in cells from ABCA1-/- mice and from Tangier individuals as well as ABCA1-expressing HeLa cells. Phospholipid movements in either normal or apoptotic lymphocytes or in macrophages were not inhibited when cells from knockout and wildtype mice or immortalized cells from Tangier individuals vs normal individuals were compared. Exposure of PS on the surface of normal thymocytes, apoptotic thymocytes and elicited peritoneal macrophages from wildtype and knockout mice or B lymphocytes from normal and Tangier individuals, as measured by annexin V binding, was also unchanged. No evidence was found of ABCA1-stimulated active PS export, and spontaneous PS movement to the outer leaflet in the presence or absence of apoA1 was unaffected by the presence or absence of ABCA1. Normal or Tangier B lymphocytes and macrophages were also identical in their ability to serve as targets or phagocytes, respectively, in apoptotic cell clearance assays. No evidence was found to support the suggestion that ABCA1 is involved in transport to the macrophage cell surface of annexins I and II, known to enhance phagocytosis of apoptotic cells. These results show that mutations in ABCA1 do not measurably reduce the rate of transbilayer movements of phospholipids in either the engulfing macrophage or the apoptotic target, thus discounting catalysis of transbilayer movements of phospholipids as the mechanism by which ABCA1 facilitates loading of phospholipids and cholesterol onto apoA1.

Aminophospholipid translocase TAT-1 promotes phosphatidylserine exposure during C. elegans apoptosis.

Phospholipids are distributed asymmetrically across the plasma-membrane bilayer of eukaryotic cells: Phosphatidylserine (PS), phosphatidylethanolamine, and phosphoinositides are predominantly restricted to the inner leaflet, whereas phophatidylcholine and sphingolipids are enriched on the outer leaflet [1, 2]. Exposure of PS on the cell surface is a conserved feature of apoptosis and plays an important role in promoting the clearance of apoptotic cells by phagocytosis [3]. However, the molecular mechanism that drives PS exposure remains mysterious. To address this issue, we studied cell-surface changes during apoptosis in the nematode C. elegans. Here, we show that PS exposure can readily be detected on apoptotic C. elegans cells. We generated a transgenic strain expressing a GFP::Annexin V reporter to screen for genes required for this process. Although none of the known engulfment genes was required, RNAi knockdown of the putative aminophospholipid transporter gene tat-1 abrogated PS exposure on apoptotic cells. tat-1(RNAi) also reduced the efficiency of cell-corpse clearance, suggesting that PS exposure acts as an "eat-me" signal in worms. We propose that tat-1 homologs might also play an important role in PS exposure in mammals.

Lipid specific activation of the murine P4-ATPase Atp8a1 (ATPase II).

The asymmetric transbilayer distribution of phosphatidylserine (PS) in the mammalian plasma membrane and secretory vesicles is maintained, in part, by an ATP-dependent transporter. This aminophospholipid "flippase" selectively transports PS to the cytosolic leaflet of the bilayer and is sensitive to vanadate, Ca(2+), and modification by sulfhydryl reagents. Although the flippase has not been positively identified, a subfamily of P-type ATPases has been proposed to function as transporters of amphipaths, including PS and other phospholipids. A candidate PS flippase ATP8A1 (ATPase II), originally isolated from bovine secretory vesicles, is a member of this subfamily based on sequence homology to the founding member of the subfamily, the yeast protein Drs2, which has been linked to ribosomal assembly, the formation of Golgi-coated vesicles, and the maintenance of PS asymmetry. To determine if ATP8A1 has biochemical characteristics consistent with a PS flippase, a murine homologue of this enzyme was expressed in insect cells and purified. The purified Atp8a1 is inactive in detergent micelles or in micelles containing phosphatidylcholine, phosphatidic acid, or phosphatidylinositol, is minimally activated by phosphatidylglycerol or phosphatidylethanolamine (PE), and is maximally activated by PS. The selectivity for PS is dependent upon multiple elements of the lipid structure. Similar to the plasma membrane PS transporter, Atp8a1 is activated only by the naturally occurring sn-1,2-glycerol isomer of PS and not the sn-2,3-glycerol stereoisomer. Both flippase and Atp8a1 activities are insensitive to the stereochemistry of the serine headgroup. Most modifications of the PS headgroup structure decrease recognition by the plasma membrane PS flippase. Activation of Atp8a1 is also reduced by these modifications; phosphatidylserine-O-methyl ester, lysophosphatidylserine, glycerophosphoserine, and phosphoserine, which are not transported by the plasma membrane flippase, do not activate Atp8a1. Weakly translocated lipids (PE, phosphatidylhydroxypropionate, and phosphatidylhomoserine) are also weak Atp8a1 activators. However, N-methyl-phosphatidylserine, which is transported by the plasma membrane flippase at a rate equivalent to PS, is incapable of activating Atp8a1 activity. These results indicate that the ATPase activity of the secretory granule Atp8a1 is activated by phospholipids binding to a specific site whose properties (PS selectivity, dependence upon glycerol but not serine, stereochemistry, and vanadate sensitivity) are similar to, but distinct from, the properties of the substrate binding site of the plasma membrane flippase.

Phosphatidylserine expression and phagocytosis of apoptotic thymocytes during differentiation of monocytic cells.

Expression of phosphatidylserine (PS) on the surface of both macrophages and their apoptotic targets is required for efficient phagocytosis. Monocytes, the precursors of macrophages, do not express PS on their surface and do not efficiently phagocytose apoptotic cells. We report here that PS appears on the surface of both human monocytic U937 cells and primary human monocytes as they differentiate in culture and acquire the ability to phagocytose apoptotic thymocytes. Phagocytosis was blocked by pretreating either the apoptotic target or the phagocyte with annexin V to mask PS and was CD14-dependent. Expression of PS, like other events characteristic of differentiating monocytes such as Mac-1 expression, was independent of the agent used to induce differentiation and was insensitive to the addition of caspase inhibitors. These results demonstrate that PS is expressed on monocytes as part of their differentiation program and is independent of apoptosis.

Reanalysis of ATP11B, a type IV P-type ATPase.

P-type ATPases are a venerable family of ATP-dependent ion transporters. Recently, evidence was presented that a rabbit gene in the type IV subfamily of P-type ATPases was missing a transmembrane helix (transmembrane domain 4) thought to be critical for ion transport, a deletion that would place the two major catalytic loops of the enzyme on opposite sides of the membrane. It was proposed that the resulting protein was a RING finger-binding protein that targets transcription factors to specific domains within the nucleus. From analysis of human genomic sequence data, it is shown here that the region containing transmembrane domain 4, corresponding to exon 12, is present in the human homolog of the gene, ATP11B. PCR analysis indicates that the predominant Atp11b transcripts in a rabbit cDNA library and in a mouse cDNA library also contain exon 12. The results suggest that the transcript proposed to encode the RING finger-binding protein is a minor rabbit-specific splice variant. The ATP11B gene thus may not encode a protein with a function radically different from that of other P-type ATPase transporters.