Margaret S. Halleck

A subfamily of P-type ATPases with aminophospholipid transporting activity

The appearance of phosphatidylserine on the surface of animal cells triggers phagocytosis and blood coagulation. Normally, phosphatidylserine is confined to the inner leaflet of the plasma membrane by an aminophospholipid translocase, which has now been cloned and sequenced. The bovine enzyme is a member of a previously unrecognized subfamily of P-type adenosine triphosphatases (ATPases) that may have diverged from the primordial enzyme before the separation of the known families of ion-translocating ATPases. Studies in Saccharomyces cerevisiae suggest that aminophospholipid translocation is a general function of members of this family.

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.

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.

Characterization of protein kinases in mitotic and meiotic cell extracts

A number of protein kinases have been separated and identified in extracts from mitotic and interphase culture cells and from mature and immature amphibian oocytes using nondenaturing polyacrylamide gel electrophoresis followed by in situ phosphorylation assays. Certain of these protein kinase activities appear to correlate with the biological activity of extracts, assayed by their ability to induce meiotic maturation following injection into Xenopus oocytes. These results are consistent with the notion that protein phosphorylation/dephosphorylation may be integral to the mechanisms of both nuclear membrane breakdown and chromosome condensation, events common and distinctive to mitosis and meiosis.

A widely distributed putative mammalian transcriptional regulator containing multiple paired amphipathic helices, with similarity to yeast SIN3

The mammalian Sin3 gene (mSin3) encodes four paired amphipathic helix (PAH) motifs, three of which and an extended region beyond PAH3 share between 59 and 70% sequence similarity with the yeast transcriptional regulator, SIN3. However, mSin3/SIN3 fusion proteins were not able to substitute for the yeast molecule in complementation assays. Transcripts encoding this putative transcriptional regulator, which maps to human chromosome 15q24, were detected in multiple mouse tissues, with highest levels seen in testis, lung, and thymus. Its wide tissue distribution suggests that mSin3, like yeast SIN3, may regulate the transcription of multiple genes.

Injected mitotic extracts induce condensation of interphase chromatin.

Although extracts from mitotic cells have been shown to induce chromosome condensation when injected into amphibian oocytes, they have not as yet been shown to induce this response in somatic interphase cells. In the experiments reported here, when mitotic extracts were injected into syncytial frog embryos, whose somatic nuclei were arrested in interphase, chromosome condensation was observed. The inability of interphase extracts, injected at similar concentrations, to induce this event demonstrates the cell cycle-specific accumulation of the factors responsible.

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 predominantAtp11b 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.

C-banding of Peromyscus constitutive heterochromatin persists following histone hyperacetylation.

Approx. 35% of the DNA of cultured cells from the cactus mouse, Peromuscus eremicus, is contained in highly condensed constitutive heterochromatin which can be visualized in metaphase chromosomes stained by the C-band technique. Previous studies have shown this constitutive heterochromatin to contain a large proportion of underacetylated, arginine-rich histones, the majority of which can be hyperacetylated when cells are treated with butyrate. In order to determine whether this simulation of the acetylated state of euchromatin alters the cytological properties of constitutive heterochromatin as well, chromosomes from butyrate-treated cells have been examined. Because of the paucity of cells in butyrate-treated cultures, prematurely condensed chromosomes (PCCs) were produced from butyrate-treated cells by fusion with mitotic cells. In these PCCs, both the highly condensed nature and the ability to C-band were preserved in the hyperacetylated constitutive heterochromatin, suggesting that the subset of arginine-rich histones which is refractory to acetylation in the presence of butyrate may be responsible for the maintenance of the heterochromatic state. In addition, PCC analyses indicated that butyrate arrests Peromyscus cells in both the G1 and G2 phases of the cell cycle and confirmed the late-replicating pattern of constitutive heterochromatin.