Circulating tumour cells--a bona fide cause of metastatic cancer.

Circulating tumour cells (CTCs) are emerging as important prognostic markers and have potential clinical utility as tumour biomarkers for targeted cancer therapy. Although CTCs were proposed more than 100 years ago as potential precursors that may form metastatic lesions, formal evidence that CTCs are indeed capable of initiating metastases is limited. Moreover, the process of CTCs shedding into the circulation, relocating to distant organ sites and initiating metastatic foci is complex and intrinsically inefficient. To partially explain the metastatic process, the concepts of CTCs as metastatic precursors or pre-metastatic conditioners have been proposed; however, it is questionable as to whether these are both variable pathways to metastasis or just markers of metastatic burden. This review explores the evidence for CTCs in the initiation and progression of metastatic cancer and the data supporting these different concepts in an attempt to better understand the role of CTCs in metastasis. A greater understanding of the metastatic potential of CTCs will open new avenues for therapeutic interventions in the future.

Type II phospholipase A2 in human gestational tissues: extractable immuno- and enzymatic activity in fetal membranes.

In this study, we have established the presence of immunoreactive (ir) Type II PLA2 in human amnion and choriodecidua obtained from women at term prior to the onset of labour. The content of irType II PLA2 present in 1 M NaCl extracts of choriodecidua and amnion averaged 3.5 +/- 3.1 and 10.6 +/- 5.2 ng/mg tissue protein (n = 3), respectively. PLA2 enzymatic activity present in the same tissues averaged 1.3 +/- 0.2 and 1.9 +/- 0.7 nmol phosphatidylethanolamine (PE) hydrolysed/mg tissue protein per h (n = 3), respectively. To allow intra-patient comparison of the relative distribution in gestational tissues, irType II PLA2 and PLA2 enzymatic activity was also determined in placenta obtained from the same group of women, and averaged 26.0 +/- 7.0 ng/mg tissue protein and 3.5 +/- 1.0 nmol PE hydrolysed/mg protein per h (n = 3), respectively. As has been previously reported for human placenta, the recovery of Type II PLA2 and PLA2 enzymatic activity from amnion and choriodecidua was increased between 16- and 25-fold when tissues were homogenized in high-ionic strength media (i.e., 10% (w/v) ammonium sulphate or 1 M NaCl) compared with that recovered when tissues were homogenized in low-ionic strength media (i.e., 0.32 M sucrose-20 mM Hepes). The data obtained represent the first quantitative estimates of immunoreactive Type II PLA2 in human amnion and choriodecidua, and support the conclusion that previous analyses of the PLA2 enzymatic activity present in gestational tissues have essentially excluded the contribution made by this PLA2 isozyme to net enzymatic activity. We suggest that this isozyme represents a major component of the PLA2 enzymatic activity present in human gestational tissues at term and that it contributes significantly to the phospholipid metabolism and arachidonic acid release which occurs during late pregnancy and at the time of labour.

Type II phospholipase A2 in human gestational tissues: subcellular distribution of placental immuno- and catalytic activity.

The aims of this study were to determine the subcellular distribution of Type II phospholipase A2 immunoactivity (irPLA2) and in vitro net PLA2 catalytic activity in human term placenta and to establish the efficacy of previously utilised homogenisation procedures with respect to the quantitative recovery of Type II PLA2 immunoreactive and in vitro net PLA2 catalytic activity. Type II PLA2 immunoactivity and PLA2 catalytic activity recovered in 900 x g supernates prepared from placental tissue (n = 3) homogenised in low ionic strength media (sucrose 0.32 M Hepes 20 mM; phosphate-buffered saline or phosphate-buffered saline containing 3 mM EGTA) was less than 10% of that recovered following homogenisation in high ionic strength medium (ammonium sulphate 10%, w/v). The subcellular distribution of Type II PLA2 immunoactivity and PLA2 catalytic activity was established by the differential centrifugation (10,000, 20,000 and 100,000 x g) of placental homogenates (n = 3). Although Type II PLA2 immunoactivity was equally distributed throughout the particulate subcellular fractions examined, PLA2 catalytic activity increased by comparison in 100,000 x g particulate material. This apparent dissociation between irType II PLA2 and catalytic activity may indicate the presence of other types of PLA2 in this fraction. The data obtained in this study indicate that previous studies which have utilised low ionic strength extractions of human gestational tissue to characterise PLA2 catalytic activity and subcellular distribution have largely excluded the contribution made by Type II PLA2. Consequently, much of the available published data on the role of PLA2 in human parturition is inadequate. A reappraisal of this enzyme's contribution to the biochemical events associated with human pregnancy and labour is required.

Functional coupling and differential regulation of the phospholipase A2-cyclooxygenase pathways in inflammation.

Prostaglandins generated by the phospholipase A2 (PLA2)/cyclooxygenase (COX) pathway are well known to mediate diverse intracellular and extracellular effects that regulate mammalian development, vascular function, renal physiology, parturition, and immune responses to infection or wounding. In immune-mediated diseases and in certain cancers, this pathway is aberrantly up-regulated and excessive prostaglandin production contributes to the pathology. It is now known that there are two isoforms of COX and multiple secreted and intracellular PLA2 enzymes. The use of isoform-specific inhibitors, coupled with antisense and in vivo gene deletion experiments, has identified independent pathways of arachidonic acid metabolism, which are differentially regulated at the levels of gene expression, protein phosphorylation, and cellular localization. There is cross-talk between the pathways at the level of PLA2 and substrate supply to the two isoforms of COX is apparently compartmentalized. Knockout studies have shown that the two COX isoforms play independent roles in immediate and delayed agonist-induced prostaglandin synthesis. Cytosolic PLA2-alpha is essential for both responses. Inducible secreted forms of PLA2 are, as yet, not essential for either response with the exception of the in vitro murine mast cell immediate response and instances of murine macrophage prostaglandin synthesis. These enzymes amplify the delayed response and are likely to modulate the severity of immune-mediated diseases.

Isolation and characterization of Rhizobium (IC3342) genes that determine leaf curl induction in pigeon pea.

Nodulation by the Rhizobium strain IC3342 causes a leaf curl syndrome in certain tropical legumes such as pigeon pea (Cajanus cajan) (N.M. Upadhyaya, J.V.D.K. Kumar Rao, D.S. Letham, and P.J. Dart, Physiological and Molecular Plant Pathology 39:357-373, 1991). Transposon (Tn5) mutagenesis of this leaf curl-inducing (Curl+) Rhizobium strain yielded two Curl- Fix- and three Curl- Fix+ mutants. Plasmid visualization and subsequent Southern blot hybridization analyses with Tn5, nif and nod gene probes showed that the Tn5 element had inserted into the symbiotic (Sym) plasmid in three of the mutants. Restriction endonuclease analyses indicated that none of the Tn5 insertions were closely linked. Tn5-containing EcoRI fragments were cloned from each mutant and used as probes to isolate the corresponding wild-type DNA fragments from a cosmid (pLAFR3) genomic library. Fix+ and/or Curl+ phenotypes were restored in each mutant by the introduction of cosmids containing the corresponding wild-type DNA. A closely related but Curl- Rhizobium strain ANU240 was shown, by Southern hybridization, to contain conserved DNA sequences of all but one of the identified genetic regions of the Curl+ Rhizobium strain IC3342. Cosmids containing the genetic region unique to the strain IC3342, designated lcr1, conferred a Curl+ phenotype on the strain ANU240. DNA sequence analysis of the cloned lcr1 region revealed five open reading frames (ORFs). The ORF2 showed homology with the Escherichia coli regulatory gene ompR, and ORF4 showed homology with E. coli and Rhizobium meliloti regulatory genes fnr and fixK, respectively.

Sequence and organization of pobA, the gene coding for p-hydroxybenzoate hydroxylase, an inducible enzyme from Pseudomonas aeruginosa.

The only recognized gene for the metabolism of p-hydroxybenzoate in Pseudomonads (pobA) has been isolated from Pseudomonas aeruginosa to provide the DNA for mutagenesis studies of the protein product, p-hydroxybenzoate hydroxylase. Since pobA is induced by p-hydroxybenzoate to produce large amounts of enzyme, its regulation in P. aeruginosa is significant. The nucleotide sequence of pobA is presented with the derived amino acid (aa) sequence, which has only two substitutions compared to the amino acid sequence obtained from the enzyme from P. fluorescens. The derived amino acid sequence predicts that the enzyme is a single polypeptide of 394 aa residues and contains one molecule of FAD. The complete structure of the protein from P. aeruginosa can be derived by analogy from the published structure of the protein from P. fluorescens. Transcription mapping was used to determine that there is one site for the initiation of mRNA synthesis in P. aeruginosa. The presence of a putative operator in the sequence suggests primary regulation by a repressor protein which binds p-hydroxybenzoate. The ribosome-binding site permits translation of the gene in Escherichia coli at levels comparable to its production in P. aeruginosa, but it produces no detectable product in E. coli under the influence of its own promoter sequence. The promoter does not conform to the common consensus sequence of E. coli promoters. The results have identified an apparent novel promoter for P. aeruginosa, which may reflect the presence of a sigma factor required for pobA induction. Repression of expression by glucose suggests a binding site in the sequence for catabolite repression.

Structural and functional analysis of nitrogenase genes from the broad-host-range Rhizobium strain ANU240.

The genes encoding the structural components of nitrogenase, nifH, nifD and nifK, from the fast-growing, broad-host-range Rhizobium strain ANU240 have been identified and characterized. They are duplicated and linked in an operon nifHDK in both copies. Sequence analysis of the nifH gene from each copy, together with partial sequence analysis of the nifD and nifK genes, and restriction endonuclease analysis suggested that the duplication is precise. Comparison of the Fe-protein sequence from strain ANU240 with that from other nitrogen-fixing organisms revealed that, despite its broad host range and certain physiological properties characteristic of Bradyrhizobium strains, ANU240 is more closely related to the narrow-host-range Rhizobium strains than to the broad-host-range Bradyrhizobium strains. The promoter regions of both copies of the nif genes contain the consensus sequence characteristic of nif promoters, and functional analysis of the two promoters suggested that both nif operons are transcribed in nodules.

Contribution of type II phospholipase A2 to in vitro phospholipase A2 enzymatic activity in human term placenta.

Although phospholipase A2 (PLA2) enzymatic activities have been implicated in the regulation of phospholipid metabolism and eicosanoid formation in human gestational tissues, the role and contribution made by individual PLA2 isozymes has not been established. The aim of this study, therefore, was to determine the contribution made by Type II PLA2 to PLA2 enzymatic activity present in human term placenta. The experimental paradigm used to establish the contribution made by Type II PLA2 to total in vitro PLA2 enzymatic activity present in placental extracts was to remove Type II PLA2 by immunoaffinity extraction and then to quantify residual PLA2 enzymatic activity. Before immunoaffinity extraction, Type II PLA2 immunoactivity and total PLA2 enzymatic activity present in placental extracts averaged 28.0 +/- 10.0 ng/mg protein and 1040 +/- 367 pmol/h per mg protein (n = 3) respectively. After solid-phase immunoaffinity batch extraction of placental extracts, immunoreactive Type II PLA2 was not detectable by ELISA, and PLA2 enzymatic activity was decreased by 82 +/- 1% (P < 0.001). Residual (i.e. non-Type II) PLA2 enzymatic activity was further characterised by Western blot analysis and enzyme activity assay. The data obtained are consistent with a contribution by both cytosolic PLA2 and other secretory PLA2 isozymes (i.e. non-Type II) to residual PLA2 enzymatic activity. The results obtained in this study support the conclusion that Type II PLA2 is quantitatively the primary PLA2 isozyme that contributes to in vitro PLA2 enzymatic activity present in extracts of human term placenta, accounting for at least 80% of total activity. These data further support the involvement of this extracellularly active isozyme in the regulation of placental phospholipid metabolism and eicosanoid formation during late gestation.

Release of Type II phospholipase A2 immunoreactivity and phospholipase A2 enzymatic activity from human placenta.

The aim of this study was to determine whether Type II phospholipase A2 (PLA2) is released from late pregnant human placental tissue. Placental explants were incubated in vitro and the release of immunoreactive (ir) Type II PLA2 and PLA2 enzymatic activity into the medium was determined. Both irType II PLA2 and PL2 enzymatic activity accumulated in the incubation medium in a time-dependent manner (P < 0.0001). This release was not associated with a loss of cell membrane integrity, as indicated by measurement of the intracellular enzyme, lactate dehydrogenase, in the incubation medium. The concentration of irType II PLA2 and PLA2 enzyme activity present in incubation medium were significantly correlated (P < 0.01). Consistent with the hypothesis that Type II PLA2 may be store in secretory granules within human placental tissue, incubation in the presence of a membrane depolarising concentration of KCI (60 mM) caused the release of irType II PLA2 2.0-fold (P < 0.001). PLA2 enzyme activity released into the incubation medium displays biochemical characteristics consistent with those previously reported for secretory PLA2 isozymes, that is, a requirement for millimolar concentrations of calcium for optimal enzyme activity, inhibited by reducing agents, such as dithiothreitol and insensitive to heat inactivation. The data obtained in this study establish that irType PLA2 is released from term placenta, when incubated in vitro. The release of this extracellularly-active PLA2 isozyme may contribute to gestational and labour-associated increases in glycerophospholipid metabolism and prostaglandin formation.

Localization of type II phospholipase A2 messenger RNA and immunoactivity in human placenta and fetal membranes.

Although Type II phospholipase A2 (PLA2) immunoactivity has been identified in homogenates of human placenta and fetal membranes, there is a paucity of information concerning the sites of synthesis of this secreted PLA2 isozyme. The aim of this study, therefore, was to establish the cellular localization of Type II PLA2 messenger RNA (mRNA) in human term placental and fetal membranes by in situ hybridization. In addition, the co-localization of immunoreactive Type II PLA2 in gestational tissues was determined, and the effect of labour status and pre-eclampsia on immunolabelling intensity were established. Type II PLA2 mRNA was identified in all tissue sections examined and was localized principally in placental villous vasculature and mesenchymal elements of placenta, chorion and amnion. Within the vasculature, Type II PLA2 mRNA was associated with smooth muscle cells. Immunoreactive Type II PLA2 was identified in the fibroblast and spongy layers of the amnion, the fibroblast and reticular layer of the chorion, and in the mesenchymal core and trophoblasts of placental villi. Immunolabelling staining intensity was greater in placenta and chorion than that observed in amnion, however, staining intensity was unaffected by labour status. Pre-eclampsia was associated with increased immunolabelling for Type II PLA2 in placenta but not fetal membranes. The data obtained clearly established that Type II PLA2 is synthesized by multiple cell types within human gestational tissues and co-localization of Type II PLA2 mRNA and immunoreactive protein has been established. The role of Type II PLA2 in gestational tissue phospholipid metabolism and, in particular, in vascular and mesenchymal elements, has yet to be established. Possible roles for this isozyme may include, the provision of substrate for eicosanoid synthesis, maintaining cell membrane phospholipid asymmetry and prevention of clot formation within the placental vascular bed.

Inactivation of thromboxane synthase with hydroperoxy-fatty acids.

Bovine lung thromboxane synthase was immobilized on phenyl-Sepharose beads by adsorption. The immobilized enzyme was catalytically active and synthesized both TXA2 and HHT. The structure-activity relationship of several hydroperoxy fatty acids and their ability to inactivate thromboxane synthase was investigated. Millimolar quantities of hydrogen peroxide and tert-butylperoxide were required to inactivate the enzyme: whereas micromolar quantities of C18 and C20 hydroperoxy fatty acids inactivated the enzyme. Pretreatment of the enzyme with long chain hydroperoxy-fatty acids resulted in a decreased synthesis of both TXB2 and HHT.

Substrate inactivation of lung thromboxane synthase preferentially decreases thromboxane A2 production.

Bovine lung thromboxane synthase was immobilized on phenyl-Sepharose beads by adsorption. The immobilized enzyme was catalytically active and synthesized both TXA2 and HHT. The production of both products was inhibited by 1-benzylimidazole and furegrelate. Multiple additions of PGH2 dramatically reduced the ability of the enzyme to synthesize TXA2, but did not effect the synthesis of HHT. In addition, 1-benzylimidazole did not protect thromboxane synthase from inactivation with multiple additions of PGH2. When the enzyme was incubated with PGH2 in the presence of 1-benzylimidazole, the synthesis of TXA2 was inhibited. When the inhibitor was removed the enzyme had still been inactivated by PGH2 in the presence of 1-benzylimidazole. Thus the substrate inactivation of the enzyme does not require the production of TXA2. Our data suggests that the synthesis of TXA2 and HHT can be differentially inactivated and may occur at different sites on the enzyme.

Measurement of PGH synthase activity using a pan-specific monoclonal antibody.

A pan-specific monoclonal antibody that recognizes a variety of prostaglandin moieties and does not recognize arachidonic acid or the hydroxy-eicosatetraenoic acids was used to assess the general ability of a tissue to produce prostaglandins. Although this assay does not give a quantitative measure of PGH synthase activity, it does provide a sensitive and convenient means of screening a large number of samples for prostaglandin production.

Circulating phospholipase A2 activity associated with sepsis and septic shock is indistinguishable from that associated with rheumatoid arthritis.

Elevation of circulating phospholipase A2 (PLA2) activity is associated with sepsis and septic shock. Elevated levels of PLA2 activity also are seen in association with chronic inflammatory disorders such as rheumatoid arthritis. The relationship between these phospholipases is unclear. We have developed a highly specific enzyme-linked immunosorbent assay (ELISA) capable of measuring human synovial PLA2 in plasma, using monoclonal antibodies raised to recombinant synovial PLA2. This ELISA has been used to quantitate circulating PLA2 levels in patients clinically diagnosed with sepsis. These elevated levels positively correlated with the elevation seen in plasma PLA2 enzyme activity. The antibodies also have been used to purify immunoreactive PLA2 from plasma of patients with sepsis, thus enabling characterization of the purified protein by amino-terminal sequence analysis. We conclude from this study that the increase in PLA2 activity seen in association with sepsis and septic shock results from a dramatic elevation in levels of a circulating PLA2 enzyme. This inflammatory PLA2 is indistinguishable, both immunologically and chemically, from that associated with rheumatoid arthritis. Therapeutic agents directed towards inhibition of this inflammatory PLA2 enzyme may have utility in the treatment of both chronic and acute inflammatory disease.

Overlapping gene expression profiles in rheumatoid fibroblast-like synoviocytes induced by the proinflammatory cytokines interleukin-1 beta and tumor necrosis factor.

OBJECTIVE AND DESIGN: The development of therapies directed against TNF alpha and IL-1 beta has underscored the importance of these cytokines in rheumatoid arthritis (RA). In this study, oligonucleotide microarrays were used to identify novel transcriptional events mediated by TNF alpha and IL-1 beta. METHODS: In this study we have used Affymetrix U95A GeneChips representing 12,600 full-length human genes to identify transcriptional events mediated by these cytokines. Fibroblast-like synoviocytes were cultured from rheumatoid synovium from RA patients and stimulated with TNF alpha and IL-1 beta. Gene transcript levels were determined using Affymetrix U95A GeneChips representing 12,600 full-length human genes. RESULTS: A large number of differentially regulated genes were identified (1.7% of array-displayed genes for TNF alpha and 2.4% for IL-1 beta), and the validity of the array protocol was subsequently confirmed using real-time PCR. The majority of the differentially expressed genes were regulated by both TNF alpha and IL-1 beta, reflecting the distal signaling pathways shared by these cytokines. A large number of novel TNF alpha and IL-1 beta-regulated genes were identified. CONCLUSIONS: A panel of novel TNF alpha- and IL-1 beta-regulated genes was identified, and these are promising candidates for further study in relation to RA and other inflammatory diseases.

Conserved nodulation genes from the non-legume symbiont Bradyrhizobium sp. (Parasponia).

A nodulation locus from the broad-host-range, non-legume symbiont Bradyrhizobium sp. (Parasponia) strain ANU289, has been identified by hybridisation to cloned Rhizobium trifolii nodulation (nod) genes. Transfer of cloned ANU289 nod genes to R.trifolii nodulation-deficient mutants showed that the locus contains a functional homologue of the R. trifolii nodD gene. DNA sequence analysis revealed the presence of three additional genes nodA, nodB and nodC clustered adjacent to nodD. The four genes from ANU289 share substantial sequence homology with those characterised from narrow-host-range Rhizobium strains. A novel 700-bp sequence inserted between the nodD and nodABC genes encodes an open reading frame designated nodK and is oriented in the same direction as nodABC. nodKABC appear to be organized in a single transcriptional unit and nodD is oriented divergently to nodKABC. A 35-bp sequence containing the ribosome binding site for the nodD gene and an AT-rich core sequence has been identified by comparison with sequences from other Rhizobium strains and is likely to be implicated in the plant-mediated induction of nodulation gene expression.

Native peptide inhibition. Specific inhibition of type II phospholipases A2 by synthetic peptides derived from the primary sequence.

The binding of low molecular weight type II phospholipase A2 (EC) to membrane surfaces and hydrolysis of phospholipid are thought to involve the formation of a hydrophobic channel into which a single substrate molecule diffuses before cleavage. The floor and right side of the channel are provided by hydrophobic residues 2, 5, and 9 of an amphipathic amino-terminal helix. The channel is postulated to form via a conformational change in this helix and inward movement of a hydrophobic flap (residue 69 side chain). We show that the amino-terminal tryptic peptide of human type II phospholipase A2 forms a noncovalent complex with the tryptic peptide from residues 70-74 of the enzyme. Further, the 70-74-peptide sequence (FLSYK) dose-dependently inhibits phospholipid hydrolysis in a mixed micelle assay. This native peptide inhibition also occurred with type II enzymes from Crotalus durissus and Crotalus atrox, which have different amino acid sequences at the amino terminus as well as different 70-74 regions of the molecules. Despite significant conservation of tertiary structure among the enzymes, inhibition by each peptide is specific to the enzyme from which the peptide sequence is derived. We propose that these native peptides inhibit enzyme activity via a sequence-specific, noncovalent interaction with the amino-terminal residues of the enzyme, thereby preventing the conformational change on binding to the micelle interface. These experiments demonstrate a new method for specific inhibition of phospholipase A2 which, in principle, would be applicable to other biologically active polypeptides and proteins.

Retinoids increase transglutaminase activity and inhibit ornithine decarboxylase activity in Chinese hamster ovary cells and in melanoma cells stimulated to differentiate.

Transglutaminase (TGase; R-glutaminyl-peptide:amine gamma-glutamyltransferase, EC 2.3.2.13) and ornithine decarboxylase (ODCase; L-ornithine carboxy-lyase, EC 4.1.1.17) activities were measured after the addition of retinoid analogs to Chinese hamster ovary (CHO) cells released from quiescence and Cloudman S91 (CCL 53.1) mouse melanoma cells stimulated to differentiate with alpha-melanocyte-stimulating hormone (MSH, melanotropin). In both cell culture lines, we detected a biphasic increase in TGase activity and a single peak of ODCase activity within 7 hr after release or stimulation. Retinoid analogs altered the expression of the initial TGase peak in both CHO and melanoma cells. Retinol increased the activity of TGase 1 hr after release in CHO cells, and the activity remained elevated until hr 4. A broad peak of TGase activity also occurred after the addition of alpha-difluoromethylornithine, an irreversible inhibitor of ODCase, and after addition of alpha-difluoromethylornithine plus retinol. In mouse melanoma cells, retinoic acid plus MSH markedly enhanced the activity of the initial TGase peak compared to MSH alone. Retinoic acid alone also increased TGase activity biphasically in these cells without the addition of MSH. These studies suggest that retinoid effects that increase TGase activity may alter the ODCase expression in proliferation and differentiation.