The conserved Helix C region in the superfamily of interferon-a/interleukin-10-related cytokines corresponds to a high-affinity binding site for the HSP70 chaperone DnaK.

HSP70 chaperones mediate protein folding by ATP-dependent interaction with short linear peptide segments that are exposed on unfolded proteins. The mode of action of the Escherichia coli homolog DnaK is representative of all HSP70 chaperones, including the endoplasmic reticulum variant BiP/GRP78. DnaK has been shown to be effective in assisting refolding of a wide variety of prokaryotic and eukaryotic proteins, including the -helical homodimeric secretory cytokine interferon- (IFN-). We screened solid-phase peptide libraries from human and mouse IFN- to identify DnaK-binding sites. Conserved DnaK-binding sites were identified in the N-terminal half of helix B and in the C-terminal half of helix C, both of which are located at the IFN- dimer interface. Soluble peptides derived from helices B and C bound DnaK with high affinity in competition assays. No DnaK-binding sites were found in the loops connecting the -helices. The helix C DnaK-binding site appears to be conserved in most members of the superfamily of interleukin (IL)-10-related cytokines that comprises, apart from IL-10 and IFN-, a series of recently discovered small secretory proteins, including IL-19, IL-20, IL-22/IL-TIF, IL-24/MDA-7 (melanoma differentiation-associated gene), IL-26/AK155, and a number of viral IL-10 homologs. These cytokines belong to a relatively small group of homodimeric proteins with highly interdigitated interfaces that exhibit the strongly hydrophobic character of the interior core of a single-chain folded domain. We propose that binding of DnaK to helix C in the superfamily of IL-10-related cytokines may constitute the hallmark of a novel conserved regulatory mechanism in which HSP70-like chaperones assist in the formation of a hydrophobic dimeric "folding" interface.

Glucose-induced oxidative stress in mesangial cells.

Background: Hyperglycaemia is a well recognized pathogenic factor of long term complications in diabetes mellitus. Hyperglycaemia not only generates reactive oxygen species but also attenuates antioxidant mechanisms creating a state of oxidative stress. Methods: Porcine mesangial cells were cultured in high glucose (HG) for ten days to investigate the effects on the antioxidant defences of the cell. Results: Mesangial cells cultured in HG conditions had significantly reduced levels of glutathione (GSH) compared with those grown in normal glucose (NG). The reduced GSH levels were accompanied by decreased gene expression of both subunits of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme in de novo synthesis of GSH. Elevated levels of intracellular malondialdehyde (MDA) were found in cells exposed to HG conditions. HG also caused elevated mRNA levels of the antioxidant enzymes CuZn superoxide dismutase (SOD) and MnSOD. These changes were accompanied by increased mRNA levels of extracellular matrix proteins (ECM), fibronectin (FN) and collagen IV (CIV). Addition of antioxidants to high glucose caused a significant reversal of FN and CIV gene expression; alpha-lipoic acid also upregulated gamma-GCS gene expression and restored intracellular GSH and MDA levels. Conclusions: We have demonstrated the existence of glucose induced-oxidative stress in mesangial cells as evidenced by elevated MDA and decreased GSH levels. The decreased levels of GSH are as a result of decreased mRNA expression of gamma-GCS within the cell. Antioxidants caused a significant reversal of FN and CIV gene expression suggesting an aetiological link between oxidative stress and increased ECM protein synthesis.

Cortactin underpins CD44-promoted invasion and adhesion of breast cancer cells to bone marrow endothelial cells

Using a validated tetracycline (tet)-regulated MCF7-founder (MCF7F) expression system to modulate expression of CD44 standard form (CD44s), we report the functional importance of CD44s and that of a novel transcriptional target of hyaluronan (HA)/CD44s signaling, EMS1/cortactin, in underpinning breast cancer metastasis. In functional experiments, tet-regulated induction of CD44s potentiated the migration and invasion of MCF7F cells through HA-supplemented Matrigel. EMS1/cortactin was identified by expression profiling as a novel transcriptional target of HA/CD44 signaling, an association validated by quantitative PCR and immunoblotting experiments in a range of breast cancer cell lines. The mechanistic basis underpinning CD44-promoted transcription of EMS1/cortactin was shown to be dependent upon a NFB mechanism, since pharmacological inhibition of IKinase-2 or suppression of p65 Rel A expression attenuated CD44-induced increases in cortactin mRNA transcript levels. Overexpression of a c-myc tagged murine cortactin construct in the weakly invasive, CD44-deficient MCF7F and T47D cells potentiated their invasion. Furthermore, the functional importance of cortactin to CD44s-promoted metastasis was demonstrated by selective suppression of cortactin in CD44-expressing MCF7F-B5 and MDA-MB-231 breast cancer cells using RNAi, which was shown to result in attenuated CD44-promoted invasion and CD44-promoted adhesion to bone marrow endothelial cells (BMECs).

Towards agent-oriented model-driven architecture

Model Driven Architecture supports the transformation from reusable models to executable software. Business representations, however, cannot be fully and explicitly represented in such models for direct transformation into running systems. Thus, once business needs change, the language abstractions used by MDA (e.g. Object Constraint Language / Action Semantics), being low level, have to be edited directly. We therefore describe an Agent-oriented Model Driven Architecture (AMDA) that uses a set of business models under continuous maintenance by business people, reflecting the current business needs and being associated with adaptive agents that interpret the captured knowledge to behave dynamically. Three contributions of the AMDA approach are identified: 1) to Agent-oriented Software Engineering, a method of building adaptive Multi-Agent Systems; 2) to MDA, a means of abstracting high level business-oriented models to align executable systems with their requirements at runtime; 3) to distributed systems, the interoperability of disparate components and services via the agent abstraction.

Agelastatin A: a novel inhibitor of osteopontin-mediated adhesion, invasion, and colony formation

Effective inhibitors of osteopontin (OPN)-mediated neoplastic transformation and metastasis are still lacking. (-)-Agelastatin A is a naturally occurring oroidin alkaloid with powerful antitumor effects that, in many cases, are superior to cisplatin in vitro. In this regard, past comparative assaying of the two agents against a range of human tumor cell lines has revealed that typically (-)-agelastatin A is 1.5 to 16 times more potent than cisplatin at inhibiting cell growth, its effects being most pronounced against human bladder, skin, colon, and breast carcinomas. In this study, we have investigated the effects of (-)-agelastatin A on OPN-mediated malignant transformation using mammary epithelial cell lines. Treatment with (-)-agelastatin A inhibited OPN protein expression and enhanced expression of the cellular OPN inhibitor, Tcf-4. (-)-Agelastatin A treatment also reduced beta-catenin protein expression and reduced anchorage-independent growth, adhesion, and invasion in R37 OPN pBK-CMV and C9 cell lines. Similar effects were observed in MDA-MB-231 and MDA-MB-435s human breast cancer cell lines exposed to (-)-agelastatin A. Suppression of Tcf-4 by RNA interference (short interfering RNA) induced malignant/invasive transformation in parental benign Rama 37 cells; significantly, these events were reversed by treatment with (-)-agelastatin A. Our study reveals, for the very first time, that (-)-agelastatin A down-regulates beta-catenin expression while simultaneously up-regulating Tcf-4 and that these combined effects cause repression of OPN and inhibition of OPN-mediated malignant cell invasion, adhesion, and colony formation in vitro. We have also shown that (-)-agelastatin A inhibits cancer cell proliferation by causing cells to accumulate in the G(2) phase of cell cycle.

Malondialdehyde scavenging and aldose-derived Schiff bases' transglycation properties of synthetic histidyl-hydrazide carnosine analogs.

Second-generation carnosine analogs bearing the histidyl-hydrazide moiety have been synthesized and tested for their efficiency in scavenging malondialdehyde (MDA) derived from lipid peroxidation and for their ability to reverse the glycation process in the glucose-ethylamine Schiff base model. The data obtained indicate that this class of compounds maintains the activity profile of carnosine and is a suitable candidate for the treatment of disorders caused by oxidative stress.

FKBPL and Peptide Derivatives: Novel Biological Agents That Inhibit Angiogenesis by a CD44-Dependent Mechanism

Purpose: Antiangiogenic therapies can be an important adjunct to the management of many malignancies. Here we investigated a novel protein, FKBPL, and peptide derivative for their antiangiogenic activity and mechanism of action.

Experimental Design: Recombinant FKBPL (rFKBPL) and its peptide derivative were assessed in a range of human microvascular endothelial cell (HMEC-1) assays in vitro. Their ability to inhibit proliferation, migration, and Matrigel-dependent tubule formation was determined. They were further evaluated in an ex vivo rat model of neovascularization and in two in vivo mouse models of angiogenesis, that is, the sponge implantation and the intravital microscopy models. Antitumor efficacy was determined in two human tumor xenograft models grown in severe compromised immunodeficient (SCID) mice. Finally, the dependence of peptide on CD44 was determined using a CD44-targeted siRNA approach or in cell lines of differing CD44 status.

Results: rFKBPL inhibited endothelial cell migration, tubule formation, and microvessel formation in vitro and in vivo. The region responsible for FKBPL's antiangiogenic activity was identified, and a 24-amino acid peptide (AD-01) spanning this sequence was synthesized. It was potently antiangiogenic and inhibited growth in two human tumor xenograft models (DU145 and MDA-231) when administered systemically, either on its own or in combination with docetaxel. The antiangiogenic activity of FKBPL and AD-01 was dependent on the cell-surface receptor CD44, and signaling downstream of this receptor promoted an antimigratory phenotype.

Conclusion: FKBPL and its peptide derivative AD-01 have potent antiangiogenic activity. Thus, these agents offer the potential of an attractive new approach to antiangiogenic therapy.

A modified Tat peptide for selective intracellular delivery of macromolecules

Objectives The Tat peptide has been widely used for the intracellular delivery of macromolecules. The aim of this study was to modify the peptide to enable regulation of cellular uptake through a dependency on activation by proteases present in the local environment.

Methods The native Tat peptide sequence was altered to inhibit the initial interaction of the peptide with the cell membrane through the addition of the consensus sequence for urokinase plasminogen activator (uPA). uPA expression was characterised and semi-quantitatively rated in three cell lines (U251mg, MDA-MB-231 and HeLa). The modified peptide was incubated with both recombinant enzyme and with cells varying in uPA activity. Cellular uptake of the modified Tat peptide line was compared with that of the native peptide and rated according to uPA activity measured in each cell line.

Key findings uPA activity was observed to be high in U251mg and MDA-MB-231 and low in HeLa. In MDA-MB-231 and HeLa, uptake of the modified peptide correlated with the level of uPA expression detected (93 and 52%, respectively). In U251mg, however, the uptake of the modified peptide was much less (19% observed reduction) than the native peptide despite a high level of uPA activity detected.

Conclusions Proteolytic activation represents an interesting strategy for the targeted delivery of macromolecules using peptide-based carriers and holds significant potential for further exploitation.

Overexpression of endoplasmic reticulum protein 29 regulates mesenchymal-epithelial transition and suppresses xenograft tumor growth of invasive breast cancer cells

Endoplasmic reticulum protein 29 (ERp29) is a novel endoplasmic reticulum ( ER) secretion factor that facilitates the transport of secretory proteins in the early secretory pathway. Recently, it was found to be overexpressed in several cancers; however, little is known regarding its function in breast cancer progression. In this study, we show that the expression of ERp29 was reduced with tumor progression in clinical specimens of breast cancer, and that overexpression of ERp29 resulted in G(0)/G(1) arrest and inhibited cell proliferation in MDA-MB-231 cells. Importantly, overexpression of ERp29 in MDA-MB-231 cells led to a phenotypic change and mesenchymal-epithelial transition (MET) characterized by cytoskeletal reorganization with loss of stress fibers, reduction of fibronectin (FN), reactivation of epithelial cell marker E-cadherin and loss of mesenchymal cell marker vimentin. Knockdown of ERp29 by shRNA in MCF-7 cells reduced E-cadherin, but increased vimentin expression. Furthermore, ERp29 overexpression in MDA-MB-231 and SKBr3 cells decreased cell migration/invasion and reduced cell transformation, whereas silencing of ERp29 in MCF-7 cells enhanced cell aggressive behavior. Significantly, expression of ERp29 in MDA-MB-231 cells suppressed tumor formation in nude mice by repressing the cell proliferative index (Ki-67 positivity). Transcriptional profiling analysis showed that ERp29 acts as a central regulator by upregulating a group of genes with tumor suppressive function, for example, E-cadherin (CDH1), cyclin-dependent kinase inhibitor (CDKN2B) and spleen tyrosine kinase (SYK), and by downregulating a group of genes that regulate cell proliferation (eg, FN, epidermal growth factor receptor ( EGFR) and plasminogen activator receptor ( uPAR)). It is noteworthy that ERp29 significantly attenuated the overall ERK cascade, whereas the ratio of p-ERK1 to p-ERK2 was highly increased. Taken together, our results showed that ERp29 is a novel regulator leading to cell growth arrest and cell transition from a proliferative to a quiescent state, and reprogramming molecular portraits to suppress the tumor growth of MDA-MB-231 breast cancer cells. Laboratory Investigation (2009) 89, 1229-1242; doi: 10.1038/labinvest.2009.87; published online 21 September 2009

Targets of genome copy number reduction in primary breast cancers identified by integrative genomics

The identification of specific oncogenes and tumor suppressor genes in regions of recurrent aneuploidy is a major challenge of molecular cancer research. Using both oligonucleotide single-nucleotide polymorphism and mRNA expression arrays, we integrated genomic and transcriptional information to identify and prioritize candidate cancer genes in regions of increased and decreased chromosomal copy number in a cohort of primary breast cancers. Confirming the validity of this approach, several regions of previously-known copy number (CN) alterations in breast cancer could be successfully reidentified. Focusing on regions of decreased CN, we defined a prioritized list of eighteen candidate genes, which included ARPIN, FBNI, and LZTSI, previously shown to be associated with cancers in breast or other tissue types, and novel genes such as P29, MORF4LI, and TBCID5. One such gene, the RUNX3 transcription factor, was selected for further study. We show that RUNX3 is present at reduced CNs in proportion to the rest of the tumor genome and that RUNX3 CN reductions can also be observed in a breast cancer series from a different center. Using tissue microarrays, we demonstrate in an independent cohort of over 120 breast tissues that RUNX3 protein is expressed in normal breast epithelium but not fat and stromal tissue, and widely down-regulated in the majority of breast cancers (> 85%). In vitro, RUNX3 overexpression suppressed the invasive potential of MDA-MB-231 breast cancer cells in a matrigel assay. Our results demonstrate the utility of integrative genomic approaches to identify novel potential cancer-related genes in primary tumors. This article contains Supplementary Material available at http:// www.interscience.wiley.com/jpages/1045-2257/suppmat. (c) 2006 Wiley-Liss, Inc.

A novel small-molecule inhibitor of IL-6 signalling.

A small library of pyrrolidinesulphonylaryl molecules has been synthesized via an efficient 4-step route, and members evaluated for their ability to inhibit IL-6 signalling. One molecule (6a) was found to have promising activity against IL-6/STAT3 signalling at the low micromolar level, and to selectively inhibit phosphorylation of STAT3 (but not STAT1) in IL-6 stimulated MDA-MB-231 breast cancer and HeLa cell lines. It was also selectively cytostatic in MDA-MB-231 (STAT3-dependent) versus A4 (STAT3-null) cells suggesting STAT3-specific inhibitory properties.

Wnt-β-catenin-Tcf-4 signalling-modulated invasiveness is dependent on osteopontin expression in breast cancer.

Background:We have previously demonstrated that Tcf-4 regulates osteopontin (OPN) in rat breast epithelial cells, Rama37. In this report, we have examined the importance of this regulation in human breast cancer.Methods:The regulatory roles of Tcf-4 on cell invasion and OPN expression were investigated. The mRNA expression of Tcf-4 and OPN, and survival of breast cancer patients were correlated.Results:Tcf-4 enhanced cell invasion in both MCF10AT and MDA MB 231 breast cancer cells by transcriptionally activating OPN expression. Osteopontin was activated by Wnt signalling in MDA MB 231 cells. Paradoxical results on Tcf-4-regulated OPN expression in MCF10AT (activation) and Rama37 (repression) cells were shown to be a result of differential Wnt signalling competency in MCF10AT and Rama37 cells. High levels of OPN and Tcf-4 mRNA expression were significantly associated with survival in breast cancer patients. Most importantly, Tcf-4-positive patients had a poorer prognosis when OPN was overexpressed, while OPN-negative patients had a better prognosis when Tcf-4 was overexpressed.Conclusion:Our results suggest that Tcf-4 can act as a repressor or activator of breast cancer progression by regulating OPN expression in a Wnt-dependent manner and that Tcf-4 and OPN together may be a novel prognostic indicator for breast cancer progression.

Rapid analysis of ecstasy and related phenethylamines in seized tablets by Raman spectroscopy

Raman spectroscopy with far-red excitation has been used to study seized, tableted samples of MDMA (N-methyl-3,4-methylenedioxyamphetamine) and related compounds (MDA, MDEA, MBDB, 2C-B and amphetamine sulfate), as well as pure standards of these drugs. We have found that by using far-red (785 nm) excitation the level of fluorescence background even in untreated seized samples is sufficiently low that there is little difficulty in obtaining good quality data with moderate 2 min data accumulation times. The spectra can be used to distinguish between even chemically-similar substances, such as the geometrical isomers MDEA and MBDB, and between different polymorphic/hydrated forms of the same drug. Moreover, these differences can be found even in directly recorded spectra of seized samples which have been bulked with other materials, giving a rapid and non-destructive method for drug identification. The spectra can be processed to give unambiguous identification of both drug and excipients (even when more than one compound has been used as the bulking agent) and the relative intensities of drug and excipient bands can be used for quantitative or at least semi-quantitative analysis. Finally, the simple nature of the measurements lends itself to automatic sample handling so that sample throughputs of 20 samples per hour can be achieved with no real difficulty.

Cell survival responses after exposure to modulated radiation fields.

In the present study survival responses were determined in cells with differing radiosensitivity, specifically primary fibroblast (AG0-1522B), human breast cancer (MDA-MB-231), human prostate cancer (DU-145) and human glioma (T98G) cells, after exposure to modulated radiation fields delivered by shielding 50% of the tissue culture flask. A significant decrease (P < 0.05) in cell survival was observed in the shielded area, outside the primary treatment field (out-of-field), that was lower than predicted when compared to uniform exposures fitted to the linear-quadratic model. Cellular radiosensitivity was demonstrated to be an important factor in the level of response for both the in- and out-of-field regions. These responses were shown to be dependent on secretion-mediated intercellular communication, because inhibition of cellular secreted factors between the in- and out-of-field regions abrogated the response. Out-of-field cell survival was shown to increase after pretreatment of cells with agents known to inhibit factors involved in mediating radiation-induced bystander signaling (aminoguanidine, DMSO or cPTIO). These data illustrate a significant decrease in survival out-of-field, dependent upon intercellular communication, in several cell lines with varying radiosensitivity after exposure to a modulated radiation field. This study provides further evidence for the importance of intercellular signaling in modulated exposures, where dose gradients are present, and may inform the refinement of established radiobiological models to facilitate the optimization of advanced radiotherapy treatment plans.