Kathryn M. Marshall

Condensin: Architect of mitotic chromosomes

Condensin is a highly conserved pentameric complex consisting of two structural maintenance of chromosome (SMC) ATPase subunits and three auxiliary components. While initially regarded as a key driver of mitotic chromosome condensation, condensin is increasingly viewed as having a more subtle influence on chromosome architecture. The two condensin complexes are required to direct the correct folding and organization of chromosomes prior to anaphase and for keeping the chromosomes compact as they separate to the poles. This ancient complex is essential in mitosis and meiosis and has additional roles in gene regulation and DNA repair. The wide variety of biochemical and genetic tools available are gradually unravelling the numerous roles condensin plays during the cell cycle and shedding light on its mechanism of action.

Glucocorticoid-Mediated Inhibition of Angiogenic Changes in Human Endothelial Cells Is Not Caused by Reductions in Cell Proliferation or Migration

Background Glucocorticoid-mediated inhibition of angiogenesis is important in physiology, pathophysiology and therapy. However, the mechanisms through which glucocorticoids inhibit growth of new blood vessels have not been established. This study addresses the hypothesis that physiological levels of glucocorticoids inhibit angiogenesis by directly preventing tube formation by endothelial cells. Methodology/Principal Findings Cultured human umbilical vein (HUVEC) and aortic (HAoEC) endothelial cells were used to determine the influence of glucocorticoids on tube-like structure (TLS) formation, and on cellular proliferation (5-bromo-2′-deoxyuridine (BrdU) incorporation), viability (ATP production) and migration (Boyden chambers). Dexamethasone or cortisol (at physiological concentrations) inhibited both basal and prostaglandin F2α (PGF2α)-induced and vascular endothelial growth factor (VEGF) stimulated TLS formation in endothelial cells (ECs) cultured on Matrigel, effects which were blocked with the glucocorticoid receptor antagonist RU38486. Glucocorticoids had no effect on EC viability, migration or proliferation. Time-lapse imaging showed that cortisol blocked VEGF-stimulated cytoskeletal reorganisation and initialisation of tube formation. Real time PCR suggested that increased expression of thrombospodin-1 contributed to glucocorticoid-mediated inhibition of TLS formation. Conclusions/Significance We conclude that glucocorticoids interact directly with glucocorticoid receptors on vascular ECs to inhibit TLS formation. This action, which was conserved in ECs from two distinct vascular territories, was due to alterations in cell morphology rather than inhibition of EC viability, migration or proliferation and may be mediated in part by induction of thrombospodin-1. These findings provide important insights into the anti-angiogenic action of endogenous glucocorticoids in health and disease.

The anti-neoplastic and novel topoisomerase II-mediated cytotoxicity of neoamphimedine, a marine pyridoacridine.

Topoisomerase IIalpha (top2) is a target of some of the most useful anticancer drugs. All clinically approved top2 drugs act to stabilize a drug-enzyme-DNA cleavable complex. Here we report the novel top2 activity of neoamphimedine, an isomer of the marine pyridoacridine amphimedine. Neoamphimedine was cytotoxic in yeast and mammalian cell lines. Neoamphimedine exhibited enhanced toxicity in top2 over-expressing yeast cells and was toxic in every mammalian cell line tested. However, neoamphimedine did not possess enhanced toxicity in a mammalian cell line sensitive to stabilized cleavable complexes. Therefore, we hypothesized that neoamphimedine is a top2-dependent drug, whose primary mechanism of action is not the stabilization of cleavable complexes. Top2-directed activity was determined in purified enzyme systems. Neoamphimedine-induced catenation of plasmid DNA only in the presence of active top2. This catenation correlated with the ability of neoamphimedine to aggregate DNA. Catenation was also observed using a filter-binding assay and transmission electron microscopy. Catenation was confirmed when only restriction enzyme digestion could resolve the catenated plasmid complex to monomer length plasmid DNA. Neoamphimedine also showed potent anti-neoplastic activity in human xenograft tumors in athymic mice. Neoamphimedine was as effective as etoposide in mice bearing KB tumors and as effective as 9-aminocamptothecin in mice bearing HCT-116 tumors. Amphimedine did not induce DNA aggregation or catenation in vitro, nor did it display any significant anti-neoplastic activity. These results suggest that neoamphimedine has a novel top2-mediated mechanism of cytotoxicity and anticancer potential.

The conserved metalloprotease invadolysin localizes to the surface of lipid droplets

Invadolysin is a metalloprotease conserved in many different organisms, previously shown to be essential in Drosophila with roles in cell division and cell migration. The gene seems to be ubiquitously expressed and four distinct splice variants have been identified in human cells but not in most other species examined. Immunofluorescent detection of human invadolysin in cultured cells reveals the protein to be associated with the surface of lipid droplets. By means of subcellular fractionation, we have independently confirmed the association of invadolysin with lipid droplets. We thus identify invadolysin as the first metalloprotease located on these dynamic organelles. In addition, analysis of larval fat-body morphological appearance and triglyceride levels in the Drosophila invadolysin mutant suggests that invadolysin plays a role in lipid storage or metabolism.

Deoxyamphimedine, a Pyridoacridine Alkaloid, Damages DNA via the Production of Reactive Oxygen Species

Marine pyridoacridines are a class of aromatic chemicals that share an 11H-pyrido[4,3,2-mn]acridine skeleton. Pyridoacridine alkaloids display diverse biological activities including cytotoxicity, fungicidal and bactericidal properties, production of reactive oxygen species (ROS) and topoisomerase inhibition. These activities are often dependent on slight modifications to the pyridoacridine skeleton. Here we demonstrate that while structurally similar to neoamphimedine and amphimedine, the biological activity of deoxyamphimedine differs greatly. Deoxyamphimedine damages DNA in vitro independent of topoisomerase enzymes through the generation of reactive oxygen species. Its activity was decreased in low oxygen, with the removal of a reducing agent and in the presence of anti-oxidants. Deoxyamphimedine also showed enhanced toxicity in cells sensitive to single or double strand DNA breaks, consistent with the in vitro activity.

Condensin, master organizer of the genome

A fundamental requirement in nature is for a cell to correctly package and divide its replicated genome. Condensin is a mechanical multisubunit complex critical to this process. Condensin uses ATP to power conformational changes in DNA to enable to correct DNA compaction, organization, and segregation of DNA from the simplest bacteria to humans. The highly conserved nature of the condensin complex and the structural similarities it shares with the related cohesin complex have provided important clues as to how it functions in cells. The fundamental requirement for condensin in mitosis and meiosis is well established, yet the precise mechanism of action is still an open question. Mutation or removal of condensin subunits across a range of species disrupts orderly chromosome condensation leading to errors in chromosome segregation and likely death of the cell. There are divergences in function across species for condensin. Once considered to function solely in mitosis and meiosis, an accumulating body of evidence suggests that condensin has key roles in also regulating the interphase genome. This review will examine how condensin organizes our genomes, explain where and how it binds the genome at a mechanical level, and highlight controversies and future directions as the complex continues to fascinate and baffle biologists.

The C-terminal flanking peptide (CTFP) of progastrin inhibits apoptosis via a PI3-kinase-dependent pathway.

Progastrin is processed to a number of peptides including glycine-extended gastrin, amidated gastrin and the C-terminal flanking peptide (CTFP). Progastrin and gastrin-gly are pro-proliferative and anti-apoptotic in gastric and colorectal cancer cell lines. The CTFP is a major form of progastrin in the stomach and colon and stimulates proliferation. However the effect of CTFP on apoptosis has not been examined. Using the human gastric carcinoma cell line AGS we show that CTFP attenuates apoptosis through a PI3-kinase pathway by stimulating the phosphorylation of Akt leading to sustained increases in the concentrations of Bcl-xL and phosphorylated Bad protein and by reducing caspase 3 activity. The anti-apoptotic effect represents an important potential mechanism for the growth promoting action of CTFP.

Activation by zinc of the human gastrin gene promoter in colon cancer cells in vitro and in vivo

Over-expression of growth factors can contribute to the development and progression of cancer, and gastrins in particular have been implicated in accelerating the development of gastrointestinal cancers. Previously our group showed that hypoxia, cobalt chloride (a hypoxia mimetic) and zinc chloride could activate the expression of the gastrin gene in vitro. To characterise activation of the gastrin promoter by zinc ions further in vivo, TALEN technology was used to engineer a luciferase reporter construct into the endogenous human gastrin gene promoter in SW480 colon cancer cells. Gastrin promoter activity in the resultant Gast(luc) SW480 colon cancer cells was then measured by bioluminescence in cell culture and in tumour xenografts in SCID mice. Activation of intracellular signalling pathways was assessed by Western blotting. Activation of the gastrin promoter by zinc ions was concentration dependent in vitro and in vivo. Zinc ions significantly stimulated phosphorylation of ERK1/2 (MAPK pathway) but not of Akt (PI3K pathway). We conclude that the endogenous gastrin promoter is responsive to zinc ions, likely via activation of the MAPK pathway.

The C-terminal flanking peptide of progastrin induces gastric cell apoptosis and stimulates colonic cell division in vivo.

Progastrin (PG) is processed into a number of smaller peptides including amidated gastrin (Gamide), non-amidated glycine-extended gastrin (Ggly) and the C-terminal flanking peptide (CTFP). Several groups have reported that PG, Gamide and Ggly are biologically active in vitro and in vivo, and are involved in the development of gastrointestinal cancers. CTFP is bioactive in vitro but little is known of its effects in vivo. This study investigated the bioactivity of CTFP in vivo in normal tissues using gastrin deficient (GASKO) mice and in two mouse models of cancer (SCID mice bearing xenograft tumors expressing normal or knocked-down levels of gastrin and a mouse model of hepatic metastasis). As with Ggly, CTFP treatment stimulated colonic proliferation in GASKO mice compared to control. CTFP also significantly increased apoptosis in the gastric mucosa of male GASKO mice. CTFP did not appear to effect xenograft growth or the incidence of liver metastases. This is the first demonstration that CTFP has specific biological activity in vivo in the colon and stomach.

Complexes of gastrin with In 3+ , Ru 3+ or Ga 3+ ions are not recognised by the cholecystokinin 2 receptor

The peptide hormone gastrin (Gamide) binds trivalent metal ions, including indium (In), ruthenium (Ru) and gallium (Ga), with high affinity. Complexes of gastrin with chelated isotopes of In and Ga have previously been used for the location of tumours expressing the cholecystokinin 2 receptor (CCK2R). The aim of the present study was to purify the complexes of Gamide with radioactive isotopes of In, Ru or Ga and to investigate their ability to bind to the CCK2R. The radioactive Gamide complexes were purified on Sep-Pak C18 cartridges or by anion exchange HPLC. Binding to the CCK2R was assessed with a stably transfected clone of the gastric carcinoma cell line AGS. The 106Ru-Gamide complex could be eluted from the C18 cartridge; the 111In-Gamide and 68Ga-Gamide complexes bound irreversibly. All three complexes were successfully purified by anion exchange HPLC. The failure to detect binding of the 111In-Gamide, 106Ru-Gamide and 68Ga-Gamide complexes to the CCK2R suggests that formation of these complexes will not be useful for the detection of tumours expressing this receptor, but may instead provide alternative ways to block the actions of Gamide as a growth factor or a stimulant of gastric acid secretion.Graphical abstractThe complexes between the hormone gastrin and radioactive 111In, 106Ru or 68Ga ions were purified by anion exchange HPLC using a NaCl gradient. The failure to detect binding of the complexes to the cholecystokinin 2 receptor suggests that metal ion treatment may provide novel approaches to block the biological actions of gastrin.