DNA supercoiling and relaxation by ATP-dependent DNA topoisomerases.

Bacterial DNA gyrase and the eukaryotic type II DNA topoisomerases are ATPases that catalyse the introduction or removal of DNA supercoils and the formation and resolution of DNA knots and catenanes. Gyrase is unique in using ATP to drive the energetically unfavourable negative supercoiling of DNA, an example of mechanochemical coupling: in contrast, eukaryotic topoisomerase II relaxes DNA in an ATP-requiring reaction. In each case, the enzyme-DNA complex acts as a 'gate' mediating the passage of a DNA segment through a transient enzyme-bridged double-strand DNA break. We are using a variety of genetic and enzymic approaches to probe the nature of these complexes and their mechanism of action. Recent studies will be described focusing on the role of DNA wrapping on the A2B2 gyrase complex, subunit activities uncovered by using ATP analogues and the coumarin and quinolone inhibitors, and the identification and functions of discrete subunit domains. Homology between gyrase subunits and the A2 homodimer of eukaryotic topo II suggests functional conservation between these proteins. The role of ATP hydrolysis by these topoisomerases will be discussed in regard to other energy coupling systems.     

Emerging roles of angiopoietin-like 4 in human cancer

Angiopoietin-like 4 (ANGPTL4) is best known for its role as an adipokine involved in the regulation of lipid and glucose metabolism. The characterization of ANGPTL4 as an adipokine is largely due to our limited understanding of the interaction partners of ANGPTL4 and how ANGPTL4 initiates intracellular signaling. Recent findings have revealed a critical role for ANGPTL4 in cancer growth and progression, anoikis resistance, altered redox regulation, angiogenesis, and metastasis. Emerging evidence suggests that ANGPTL4 function may be drastically altered depending on the proteolytic processing and posttranslational modifications of ANGPTL4, which may clarify several conflicting roles of ANGPTL4 in different cancers. Although the N-terminal coiled-coil region of ANGPTL4 has been largely responsible for the endocrine regulatory role in lipid metabolism, insulin sensitivity, and glucose homeostasis, it has now emerged that the COOH-terminal fibrinogen-like domain of ANGPTL4 may be a key regulator in the multifaceted signaling during cancer development. New insights into the mechanistic action of this functional domain have opened a new chapter into the possible clinical application of ANGPTL4 as a promising candidate for clinical intervention in the fight against cancer. This review summarizes our current understanding of ANGPTL4 in cancer and highlights areas that warrant further investigation. A better understanding of the underlying cellular and molecular mechanisms of ANGPTL4 will reveal novel insights into other aspects of tumorigenesis and the potential therapeutic value of ANGPTL4.     

Molecular cloning and characterization of the human topoisomerase IIalpha and IIbeta genes: evidence for isoform evolution through gene duplication

Human DNA topoisomerase II is essential for chromosome segregation and is the target for several clinically important anticancer agents. It is expressed as genetically distinct alpha and beta isoforms encoded by the TOP2alpha and TOP2beta genes that map to chromosomes 17q21-22 and 3p24, respectively. The genes display different patterns of cell cycle- and tissue-specific expression, with the alpha isoform markedly upregulated in proliferating cells. In addition to the fundamental role of TOP2alpha and TOP2beta genes in cell growth and development, altered expression and rearrangement of both genes are implicated in anticancer drug resistance. Here, we report the complete structure of the human topoisomerase IIalpha gene, which consists of 35 exons spanning 27.5 kb. Sequence data for the exon-intron boundaries were determined and examined in the context of topoisomerase IIalpha protein structure comprising three functional domains associated with energy transduction, DNA breakage-reunion activity and nuclear localization. The organization of the 3' half of human TOP2beta, including sequence specifying the C-terminal nuclear localization domain, was also elucidated. Of the 15 introns identified in this 20 kb region of TOP2beta, the first nine and the last intron align in identical positions and display the same phases as introns in TOP2alpha. Though their extreme 3' ends differ, the striking conservation suggests the two genes diverged recently in evolutionary terms consistent with a gene duplication event. Access to TOP2alpha and TOP2beta gene structures should aid studies of mutations and gene rearrangements associated with anticancer drug resistance.     

Preservation of neurological functions by nitric oxide synthase inhibitors in conscious rats following delayed hemorrhagic shock

Excessive production of nitric oxide (NO) as result of inducible nitric oxide synthase (iNOS) induction has been implicated in the pathophysiology of hemorrhagic shock. Our aim was to study the effects of NOS inhibitors, aminoguanidine (AG) and NG-nitro-L-arginine methyl ester (L-NAME), on survival rate, mean arterial blood pressure (MABP), temporal evolution of infarct volume, nitric oxide (NO) production and neurological deficit in a model of delayed hemorrhagic shock (DHS) in conscious rats. Our results showed that the NOS inhibitors significantly improved survival rate, MABP, and attenuated brain NO overproduction 24, 48 h and 72 h after DHS. AG reduced brain infarct volume and improved the neurological performance evaluated by the rotameric and grip strength tests while L-NAME did not show protective effect in rats following DHS. These findings suggest that NO formation via iNOS activation may contribute to organ damage and that the selective iNOS inhibitor, AG, may be of interest as a therapeutic agent for neurological recovery following DHS.     

Prevalence of PALB2 Mutations in Breast Cancer Patients in Multi-Ethnic Asian Population in Malaysia and Singapore

Background

The partner and localizer of breast cancer 2 (PALB2) is responsible for facilitating BRCA2-mediated DNA repair by serving as a bridging molecule, acting as the physical and functional link between the breast cancer 1 (BRCA1) and breast cancer 2 (BRCA2) proteins. Truncating mutations in the PALB2 gene are rare but are thought to be associated with increased risks of developing breast cancer in various populations.

Methods

We evaluated the contribution of PALB2 germline mutations in 122 Asian women with breast cancer, all of whom had significant family history of breast and other cancers. Further screening for nine PALB2 mutations was conducted in 874 Malaysian and 532 Singaporean breast cancer patients, and in 1342 unaffected Malaysian and 541 unaffected Singaporean women.

Results

By analyzing the entire coding region of PALB2, we found two novel truncating mutations and ten missense mutations in families tested negative for BRCA1/2-mutations. One additional novel truncating PALB2 mutation was identified in one patient through genotyping analysis. Our results indicate a low prevalence of deleterious PALB2 mutations and a specific mutation profile within the Malaysian and Singaporean populations.     

FANCE: the link between Fanconi anaemia complex assembly and activity

The Fanconi anaemia (FA) nuclear complex (composed of the FA proteins A, C, G and F) is essential for protection against chromosome breakage. It activates the downstream protein FANCD2 by monoubiquitylation; this then forges an association with the BRCA1 protein at sites of DNA damage. Here we show that the recently identified FANCE protein is part of this nuclear complex, binding both FANCC and FANCD2. Indeed, FANCE is required for the nuclear accumulation of FANCC and provides a critical bridge between the FA complex and FANCD2. Disease-associated FANCC mutants do not bind to FANCE, cannot accumulate in the nucleus and are unable to prevent chromosome breakage.