Inhibition of transforming growth factor-beta 1 signaling attenuates ataxia telanglectasia mutated activity in response to genotoxic stress

Ionizing radiation causes DNA damage that elicits a cellular program of damage control coordinated by the kinase activity of ataxia telangiectasia mutated protein (ATM). Transforming growth factor beta (TGF beta)-1, which is activated by radiation, is a potent and pleiotropic mediator of physiologic and pathologic processes. Here we show that TGF beta inhibition impedes the canonical cellular DNA damage stress response. Irradiated Tgf beta 1 nail murine epithelial cells or human epithelial cells treated with a small-molecule inhibitor of TGF beta type I receptor kinase exhibit decreased phosphorylation of Chk2, Rad17, and p53; reduced gamma H2AX radiation-induced foci; and increased radiosensitivity compared with TGF beta competent cells. We determined that loss of TGF beta signaling in epithelial cells truncated ATM autophosphorylation and significantly reduced its kinase activity, without affecting protein abundance. Addition of TGF beta restored functional ATM and downstream DNA damage responses. These data reveal a heretofore undetected critical link between the microenvironment and ATM, which directs epithelial cell stress responses, cell fate, and tissue integrity. Thus, Tgf beta 1, in addition to its role in homoeostatic growth control, plays a complex role in regulating responses to genotoxic stress, the failure of which would contribute to the development of cancer; conversely, inhibiting TGF beta may be used to advantage in cancer therapy.

Testicular germ cell tumors exhibit evidence of hormone dependence

The aim of this investigation was to test the hypothesis that testicular germ cell tumors (TGCTs) are hormone-dependent cancers. Human TGCT cells were implanted in the left testis of male severe combined immunodeficient mice receiving either no treatment or hormone manipulation treatment [blockade of gonadotropin-releasing hormone secretion and/or signaling using leuprolide or leuprolide plus exogenous testosterone]. Real-time RT-PCR analysis was used to determine the expression profiles of hormone pathway-associated genes. Tumor burden was significantly smaller in mice receiving both leuprolide and testosterone. Real-time RTPCR analysis of follicle-stimulating hormone (FSH) receptor, luteinizing hormone (LH) receptor and P450 aromatase revealed changes in expression in normal testis tissue related to presence of xenograft tumors and manipulation of hormone levels but a complete absence of expression of these genes in tumor cells themselves. This was confirmed in human specimens of TGCT. Reduced TGCT growth in vivo was associated with significant downregulation of LH receptor and P450 aromatase expression in normal testes. In conclusion, manipulation of hormone levels influenced the growth of TGCT in vivo, while the presence of xenografted tumors influenced the expression of hormone-related genes in otherwise untreated animals. Human TGCTs, both in the animal model and in clinical specimens, appear not to express receptors for FSH or LH. Similarly, expression of the P450 aromatase gene is absent in TGCTs. Impaired estrogen synthesis and/or signaling may be at least partly responsible for inhibition of TGCT growth in the animal model. (c) 2005 Wiley-Liss, Inc.

Conservation and accessibility of an inner core lipopolysaccharide epitope of Neisseria meningitidis

We investigated the conservation and antibody accessibility of inner core epitopes of Neisseria meningitidis lipopolysaccharide (LPS) because of their potential as vaccine candidates. An immunoglobulin G3 murine monoclonal antibody (MAb), designated MAb B5, was obtained by immunizing mice with a galE mutant of N. meningitidis H44/76 (B.15.P1.7,16 immunotype L3). We have shown that MAb B5 can bind to the core LPS of wild-type encapsulated MC58 (B.15.P1.7,16 immunotype L3) organisms in vitro and ex vivo. An inner core structure recognized by MAb B5 is conserved and accessible in 26 of 34 (76%) of group B and 78 of 112 (70%) of groups A, C, W, X, Y, and Z strains. N. meningitidis strains which possess this epitope are immunotypes in which phosphoethanolamine (PEtn) is linked to the 3-position of the beta-chain heptose (HepII) of the inner core. In contrast, N. neningitidis strains lacking reactivity with MAb B5 have an alternative core structure in which PEtn is linked to an exocyclic position (i.e., position 6 or 7) of HepII (immunotypes L2, L4, and L6) or is absent (immunotype L5). We conclude that MAb B5 defines one or more of the major inner core glycoforms of N. meningitidis LPS. These findings support the possibility that immunogens capable of eliciting functional antibodies specific to inner core structures could be the basis of a vaccine against invasive infections caused by N. meningitidis.

Genome-wide analysis identifies a general requirement for polarity proteins in endocytic traffic

In a genome-wide RNA-mediated interference screen for genes required in membrane traffic - including endocytic uptake, recycling from endosomes to the plasma membrane, and secretion - we identified 168 candidate endocytosis regulators and 100 candidate secretion regulators. Many of these candidates are highly conserved among metazoans but have not been previously implicated in these processes. Among the positives from the screen, we identified PAR-3, PAR-6, PKC-3 and CDC-42, proteins that are well known for their importance in the generation of embryonic and epithelial-cell polarity. Further analysis showed that endocytic transport in Caenorhabditis elegans coelomocytes and human HeLa cells was also compromised after perturbation of CDC-42/Cdc42 or PAR-6/Par6 function, indicating a general requirement for these proteins in regulating endocytic traffic. Consistent with these results, we found that tagged CDC-42/Cdc42 is enriched on recycling endosomes in C. elegans and mammalian cells, suggesting a direct function in the regulation of transport.

S100P dissociates myosin IIA filaments and focal adhesion sites to reduce cell adhesion and enhance cell migration

S100 proteins promote cancer cell migration and metastasis. To investigate their roles in the process of migration we have constructed inducible systems for S100P in rat mammary and human HeLa cells that show a linear relationship between its intracellular levels and cell migration. S100P, like S100A4, differentially interacts with the isoforms of nonmuscle myosin II (NMIIA, K(d) = 0.5 µm; IIB, K(d) = 8 µm; IIC, K(d) = 1.0 µm). Accordingly, S100P dissociates NMIIA and IIC filaments but not IIB in vitro. NMIIA knockdown increases migration in non-induced cells and there is no further increase upon induction of S100P, whereas NMIIB knockdown reduces cell migration whether or not S100P is induced. NMIIC knockdown does not affect S100P-enhanced cell migration. Further study shows that NMIIA physically interacts with S100P in living cells. In the cytoplasm, S100P occurs in discrete nodules along NMIIA-containing filaments. Induction of S100P causes more peripheral distribution of NMIIA filaments. This change is paralleled by a significant drop in vinculin-containing, actin-terminating focal adhesion sites (FAS) per cell. The induction of S100P, consequently, causes significant reduction in cellular adhesion. Addition of a focal adhesion kinase (FAK) inhibitor reduces disassembly of FAS and thereby suppresses S100P-enhanced cell migration. In conclusion, this work has demonstrated a mechanism whereby the S100P-induced dissociation of NMIIA filaments leads to a weakening of FAS, reduced cell adhesion, and enhanced cell migration, the first major step in the metastatic cascade.

Understanding the yeast host cell response to recombinant membrane protein production

Membrane proteins are drug targets for a wide range of diseases. Having access to appropriate samples for further research underpins the pharmaceutical industry's strategy for developing new drugs. This is typically achieved by synthesizing a protein of interest in host cells that can be cultured on a large scale, allowing the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to that of the native human source cells of many proteins of interest, while also being quick, easy and cheap to grow and process. Even in these cells, the production of human membrane proteins can be plagued by low functional yields; we wish to understand why. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast host strains. By relieving the bottlenecks to recombinant membrane protein production in yeast, we aim to contribute to the drug discovery pipeline, while providing insight into translational processes.

Cellular uptake and biological activity of synthetic hammerhead ribozymes

Glioblastoma Multiforme (GBM) is a highly malignant form of brain cancer for which there is no effective cure. The over-expression of a number of genes, including the epidermal growth factor receptor (EGFr), has been implicated as a causative factor of tumourigenesis. Ribozymes are a class of ribonucleic acid that possess enzymatic properties. They can inhibit gene-expression in a highly sequence specific manner by catalysing the trans-cleavage of target RNA. The potential use of synthetic hammerhead ribozymes as novel anti-brain tumour agents was investigated in this study. The successful use of synthetic, exogenously administered ribozymes for such applications will require chemical modifications that improve biological stability and a fundamental understanding of cellular uptake mechanisms. Chimeric 2'-O-methylated hammerhead ribozymes proved to be significantly more stable (>4000-fold) in serum than unmodified RNA ribozymes and exhibited high in vitro catalytic activity. The cellular association of an internally [32P]-labelled 2'-O-methylated chimeric ribozyme in U87-MG human glioma cells was temperature-, energy- and pH-dependent and involved an active process that could be competed with a variety of polyanions. Indications are that the predominant mechanism of uptake is by adsorptive and / or receptor mediated endocytosis. Twenty 2'-O-methylated chimeric ribozymes were designed to cleave various sites along the EGFr mRNA. In vitro, 18 ribozymes exhibited high activity in cleaving a complementary short substrate. Using LipofectAMINETM as a delivery agent, the efficacy of these ribozymes was evaluated in the A431 cell line, which expresses amplified levels of EGFr. Studies revealed that although the ribozymes were taken up by the cells and remained stable over a period of 4 days, no significant reduction in either EGFr expression or cell proliferation was evident. The presence of telomerase, a ribonucleoprotein responsible for telomere elongation, has been strongly associated with tumour progression. The biological activity of a 2'-O-methylated ribozyme targeted against the RNA component of telomerase was determined. The ribozyme exhibited specific dose-dependent inhibition of telomerase activity in U87-MG cell lysates with an IC50 of –4μM. When 4μM ribozyme was delivered to intact U87-MG cells, complexed to LipofectAMINETM, telomerase activity was significantly reduced to 74.5±4.17% of the untreated control. Free ribozyme showed no significant inhibitory effect demonstrating the importance of an appropriate delivery system for optimum delivery of exogenously administered ribozymes.

Methylglyoxal, glyoxalses and cell proliferation

The metabolic function of the glyoxalase system was investigated in (a) the differentiation and proliferation of human tumour cells in vitro, (b) the cell-free assembly of microtubules and (c) in the red blood cells during hyperglycaemia associated with Diabetes Mellitus. Chemically-induced differentiation of human promyelocytic HL60 leukaemia cells to neutrophils, and K562 erythroleukaemia cells, was accompanied by a decrease and an increase in the activity of glyoxalase I, respectively. Growth-arrest of Burkitt's lymphoma Raji cells and GM892 lymphoblastoid cells was accompanied by an increase and a decrease in the activity of glyoxalase I respectively. However, differentiation and growth arrest generally proceeded with an increase in the activity of glyoxalase II. Glyoxalase I activity did not consistently correlate with cell differentiation or proliferation status; hence, it is unlikely that glyoxalase I activity is either an indicator or a regulator of cell differentiation or proliferation. Conversely, glyoxalase II activity consistently increased during cell differentiation and growth-arrest and may be both an indicator and regulator of cell differentiation or proliferation. This may be related to the control of cellular microtubule assembly. S-D-Lactoylglutathione potentiated the cell-free, GTP-promoted assembly of microtubules. The effect was dose-related and was inhibited by glyoxalase II. During assembly, S-D-lactoylglutathione was consumed. This suggests that the glyoxalase system, through the influence of S-D-lactoylglutathione, may regulate the assembly of microtubules in cellular systems The whole blood concentrations of methylglyoxal and S-D-lactoylglutathione were increased in Diabetes Mellitus. There was no significant difference between red blood cell glyoxalase activities in diabetics, compared to healthy controls. However, insulin-dependent diabetic patients with retinopathy had a significantly higher glyoxalase I activity and a lower glyoxalase II activity, than patients without retinopathy. Diabetic retinopathy correlated with high glyoxalase I activity and low glyoxalase II activity and suggests the glyoxalase system may be involved in the development of diabetic complications.

Optimising yeast as a host for recombinant protein production (review)

Having access to suitably stable, functional recombinant protein samples underpins diverse academic and industrial research efforts to understand the workings of the cell in health and disease. Synthesising a protein in recombinant host cells typically allows the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to the native human source cells of many proteins of interest, while also being quick, easy, and cheap to grow and process. Even in these cells the production of some proteins can be plagued by low functional yields. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast cell factories. In this chapter, we provide an overview of the opportunities available to improve yeast as a host system for recombinant protein production.

Investigating the delivery of antimicrobial proteins and aminoglycoside antibiotics to the airways

Biopharmaceuticals are finding wide applications in the management of diverse disease conditions. Pulmonary delivery of proteins may constitute an effective and efficient non-invasive alternative to parenteral delivery, which is currently the main route of administration of biopharmaceutical drugs. A particular area, in which pulmonary delivery of peptides and proteins may find ready application, is in the local delivery of antimicrobial peptides and proteins to the airway, a measure that could potentially bring about improvements to currently available antipseudomonal therapies. This thesis has therefore sought to develop inhalable antimicrobial proteins in combination with antibiotics that have particularly good antimicrobial activity against Pseudomonas aeruginosa infections in the respiratory tract of people with cystic fibrosis (CF). Through process optimisation, a suitable spray drying method was developed and used for the preparation of active, inhalable dry powder formulations of the antimicrobial protein, lactoferrin, and aminoglycosides (tobramycin and gentamicin). The physicochemical properties, aerosolisation performance and the antibacterial properties of the various spray-dried formulations were assessed. In addition, a relevant in vitro cellular model was employed to investigate the potential cytotoxic and pro-inflammatory effects of the various formulations on four bronchial human epithelial cells together with their effectiveness at reducing bacterial colonies when administered on to biofilm co-cultured on the epithelial cells. It was found that following spray drying the particles obtained were mostly spherical, amorphous and possessed suitable aerosolisation characteristics. The various spray-dried antimicrobial proteins (lactoferrin or apo lactoferrin) and co-spray dried combinations of the proteins and aminoglycosides were found to exhibit bactericidal activity against planktonic and biofilms of P. aeruginosa. In general, the spray drying process was found not to significantly affect the antimicrobial activities of the protein. Treatment of the different bronchial epithelial cell lines with the antimicrobial formulations showed that the various formulations were non-toxic and that the co-spray dried combinations significantly reduced established P. aeruginosa biofilms on the four bronchial epithelial cells. Overall, the results from this thesis demonstrates that spray drying could potentially be employed to prepare inhalable antimicrobial agents comprised of proteins and antibiotics. These new combinations of proteins and aminoglycosides has promising applications in the management of P. aeruginosa in the airway of cystic fibrosis patients.

Meningiomas expressing and responding to cholecystokinin (CCK)

The effect of cholecystokinin (CCK) on cultured human meningioma derived cells was investigated. Exposure of meningioma cells for 6-12 days to CCK-8s (2-200 nM) resulted in a dose dependent stimulation of cell growth to a maximum of 1.1-fold over basal controls. A time course study showed stimulation of cell growth at day 3 followed by increase throughout day 6. The stimulatory effect of CCK on meningioma cell growth was completely abolished by a CCK-B specific receptor antagonist, L-365,260. Reverse-transcription of meningioma-derived RNA into cDNA followed by amplification by the polymerase chain reaction using specific primers for CCK peptide and its CCK-A and/B receptor revealed 100% presence of CCK peptide and CCK-B receptors mRNA whereas CCK-A receptor was expressed in 66% of the meningiomas. These results provide evidence that human meningioma cells possess CCK peptide and its receptors the activation of which leads to increase of cell growth possibly via an autocrine/paracrine mechanism. © Springer 2005.

Bifunctional role for VEGF-induced heme oxygenase-1 in vivo:induction of angiogenesis and inhibition of leukocytic infiltration

Heme-oxygenases (HOs) catalyze the conversion of heme into carbon monoxide and biliverdin. HO-1 is induced during hypoxia, ischemia/reperfusion, and inflammation, providing cytoprotection and inhibiting leukocyte migration to inflammatory sites. Although in vitro studies have suggested an additional role for HO-1 in angiogenesis, the relevance of this in vivo remains unknown. We investigated the involvement of HO-1 in angiogenesis in vitro and in vivo. Vascular endothelial growth factor (VEGF) induced prolonged HO-1 expression and activity in human endothelial cells and HO-1 inhibition abrogated VEGF-driven angiogenesis. Two murine models of angiogenesis were used: (1) angiogenesis initiated by addition of VEGF to Matrigel and (2) a lipopolysaccharide (LPS)-induced model of inflammatory angiogenesis in which angiogenesis is secondary to leukocyte invasion. Pharmacologic inhibition of HO-1 induced marked leukocytic infiltration that enhanced VEGF-induced angiogenesis. However, in the presence of an anti-CD18 monoclonal antibody (mAb) to block leukocyte migration, VEGF-induced angiogenesis was significantly inhibited by HO-1 antagonists. Furthermore, in the LPS-induced model of inflammatory angiogenesis, induction of HO-1 with cobalt protoporphyrin significantly inhibited leukocyte invasion into LPS-conditioned Matrigel and thus prevented the subsequent angiogenesis. We therefore propose that during chronic inflammation HO-1 has 2 roles: first, an anti-inflammatory action inhibiting leukocyte infiltration; and second, promotion of VEGF-driven noninflammatory angiogenesis that facilitates tissue repair.

Optimising yeast as a host for recombinant protein production (review)

Having access to suitably stable, functional recombinant protein samples underpins diverse academic and industrial research efforts to understand the workings of the cell in health and disease. Synthesising a protein in recombinant host cells typically allows the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to the native human source cells of many proteins of interest, while also being quick, easy, and cheap to grow and process. Even in these cells the production of some proteins can be plagued by low functional yields. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast cell factories. In this chapter, we provide an overview of the opportunities available to improve yeast as a host system for recombinant protein production.

Drug repurposing screen identifies Foxo1-dependent angiopoietin-2 regulation in sepsis

Objective: The recent withdrawal of a targeted sepsis therapy has diminished pharmaceutical enthusiasm for developing novel drugs for the treatment of sepsis. Angiopoietin-2 is an endothelial-derived protein that potentiates vascular inflammation and leakage and may be involved in sepsis pathogenesis. We screened approved compounds for putative inhibitors of angiopoietin-2 production and investigated underlying molecular mechanisms. Design: Laboratory and animal research plus prospective placebo-controlled randomized controlled trial (NCT00529139) and retrospective analysis (NCT00676897). Setting: Research laboratories of Hannover Medical School and Harvard Medical School. Patients: Septic patients/C57Bl/6 mice and human endothelial cells. Interventions: Food and Drug Administration-approved library screening. Measurements and Main Results: In a cell-based screen of more than 650 Food and Drug Administration-approved compounds, we identified multiple members of the 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor drug class (referred to as statins) that suppressed angiopoietin-2. Simvastatin inhibited 3-hydroxy-3-methyl-glutaryl-CoA reductase, which in turn activated PI3K-kinase. Downstream of this signaling, PI3K-dependent phosphorylation of the transcription factor Foxo1 at key amino acids inhibited its ability to shuttle to the nucleus and bind cis-elements in the angiopoietin-2 promoter. In septic mice, transient inhibition of angiopoietin-2 expression by liposomal siRNA in vivo improved absolute survival by 50%. Simvastatin had a similar effect, but the combination of angiopoietin-2 siRNA and simvastatin showed no additive benefit. To verify the link between statins and angiopoietin-2 in humans, we performed a pilot matched case-control study and a small randomized placebo-controlled trial demonstrating beneficial effects on angiopoietin-2. Conclusions: 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors may operate through a novel Foxo1-angiopoietin-2 mechanism to suppress de novo production of angiopoietin-2 and thereby ameliorate manifestations of sepsis. Given angiopoietin-2's dual role as a biomarker and candidate disease mediator, early serum angiopoietin-2 measurement may serve as a stratification tool for future trials of drugs targeting vascular leakage.