Phenserine regulates translation of β-amyloid precursor protein mRNA by a putative interleukin-1 responsive element, a target for drug development

The reduction in levels of the potentially toxic amyloid-β peptide (Aβ) has emerged as one of the most important therapeutic goals in Alzheimer's disease. Key targets for this goal are factors that affect the expression and processing of the Aβ precursor protein (βAPP). Earlier reports from our laboratory have shown that a novel cholinesterase inhibitor, phenserine, reduces βAPP levels in vivo. Herein, we studied the mechanism of phenserine's actions to define the regulatory elements in βAPP processing. Phenserine treatment resulted in decreased secretion of soluble βAPP and Aβ into the conditioned media of human neuroblastoma cells without cellular toxicity. The regulation of βAPP protein expression by phenserine was posttranscriptional as it suppressed βAPP protein expression without altering βAPP mRNA levels. However, phenserine's action was neither mediated through classical receptor signaling pathways, involving extracellular signal-regulated kinase or phosphatidylinositol 3-kinase activation, nor was it associated with the anticholinesterase activity of the drug. Furthermore, phenserine reduced expression of a chloramphenicol acetyltransferase reporter fused to the 5′-mRNA leader sequence of βAPP without altering expression of a control chloramphenicol acetyltransferase reporter. These studies suggest that phenserine reduces Aβ levels by regulating βAPP translation via the recently described iron regulatory element in the 5′-untranslated region of βAPP mRNA, which has been shown previously to be up-regulated in the presence of interleukin-1. This study identifies an approach for the regulation of βAPP expression that can result in a substantial reduction in the level of Aβ.

Id1 regulation of cellular senescence through transcriptional repression of p16/Ink4a

The Id family of helix–loop–helix (HLH) transcriptional regulatory proteins does not possess a basic DNA-binding domain and functions as a negative regulator of basic HLH transcription factors. Id proteins coordinate cell growth and differentiation pathways within mammalian cells and have been shown to regulate G1-S cell-cycle transitions. Although much recent data has implicated Id1 in playing a critical role in modulating cellular senescence, no direct genetic evidence has been reported to substantiate such work. Here we show that Id1-null primary mouse embryo fibroblasts undergo premature senescence despite normal growth profiles at early passage. These cells possess increased expression of the tumor-suppressor protein p16/Ink4a but not p19/ARF, and have decreased cyclin-dependent kinase (cdk) 2 and cdk4 kinase activity. We also show that Id1 is able to directly inhibit p16/Ink4a but not p19/ARF promoter activity via its HLH domain, and that Id1inhibits transcriptional activation at E-boxes within the p16/Ink4a promoter. Our data provide, to our knowledge, the first genetic evidence for a role for Id1 as an inhibitor of cellular senescence and suggest that Id1 functions to delay cellular senescence through repression of p16/Ink4a. Because epigenetic and genetic abrogation of p16/Ink4a function has been implicated in the evolution of several human malignancies, we propose that transcriptional regulation of p16/Ink4a may also provide a mechanism for the dysregulation of normal cellular growth controls during the evolution of human malignancies.

An AML-1 consensus sequence binds an osteoblast-specific complex and transcriptionally activates the osteocalcin gene.

Tissue and cell-type specific expression of the rat osteocalcin (rOC) gene involves the interplay of multiple transcriptional regulatory factors. In this report we demonstrate that AML-1 (acute myeloid leukemia-1), a DNA-binding protein whose genes are disrupted by chromosomal translocations in several human leukemias, interacts with a sequence essential for enhancing tissue-restricted expression of the rOC gene. Deletion analysis of rOC promoter-chloramphenicol acetyltransferase constructs demonstrates that an AML-1-binding sequence within the proximal promoter (-138 to -130 nt) contributes to 75% of the level of osteocalcin gene expression. The activation potential of the AML-1-binding sequence has been established by overexpressing AML-1 in osteoblastic as well as in nonosseous cell lines. Overexpression not only enhances rOC promoter activity in osteoblasts but also mediates OC promoter activity in a nonosseous human fibroblastic cell line. A probe containing this site forms a sequence specific protein-DNA complex with nuclear extracts from osteoblastic cells but not from nonosseous cells. Antisera supershift experiments indicate the presence of AML-1 and its partner protein core-binding factor beta in this osteoblast-restricted complex. Mutations of the critical AML-1-binding nucleotides abrogate formation of the complex and strongly diminish promoter activity. These results indicate that an AML-1 related protein is functional in cells of the osteoblastic lineage and that the AML-1-binding site is a regulatory element important for osteoblast-specific transcriptional activation of the rOC gene.

Glucocorticoid and progestin receptors are differently involved in the cooperation with a structural element of the mouse mammary tumor virus promoter.

We have previously characterized a regulatory element located between -294 and -200 within the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). This element termed AA element cooperates with the glucocorticoid response elements (GREs) for glucocorticoid activation. Here we show that in a MMTV LTR wild type context, the deletion of this element significantly reduces both glucocorticoid and progestin activation of the promoter. Deletion of the two most distal GREs forces the glucocorticoid receptor (GR) and the progestin receptor (PR) to bind the same response elements and results in a dramatic decrease in the inducibility of the MMTV promoter by the two hormones. The simultaneous deletion of the two distal GREs and of the AA element abolishes completely the glucocorticoid-induced activation of the promoter. In contrast it restores a significant level of progestin-induced activation. This different effect of the double deletion on glucocorticoid- and progestin-induced MMTV promoter activation is not cell specific because it is also observed, and is even stronger, when either GR or PR is expressed in the same cell line (NIH 3T3). This is the first description of a mutated MMTV promoter that, although retaining GREs, is activated by progestins and not by glucocorticoids. This suggests a different functional cooperation between protein(s) interacting with the AA element and GR or PR. Cotransfections with constructs containing wild-type or mutated MMTV LTR with either PR lacking its C-terminal domain or GR/PR chimeras in which the N-terminal domains have been exchanged demonstrate that the N-terminal domains of the receptors specify the different behavior of GR and PR regarding the AA element.

Human immunodeficiency virus type 1 and 2 Tat proteins specifically interact with RNA polymerase II.

The Tat-responsive region (TAR) element is a critical RNA regulatory element in the human immunodeficiency virus (HIV) long terminal repeat, which is required for activation of gene expression by the transactivator protein Tat. Recently, we demonstrated by gel-retardation analysis that RNA polymerase II binds to TAR RNA and that Tat prevents this binding even when Tat does not bind to TAR RNA. These results suggested that direct interactions between Tat and RNA polymerase II may prevent RNA polymerase II pausing and lead to Tat-mediated increases in transcriptional elongation. To test this possibility, we performed protein interaction studies with RNA polymerase II and both the HIV-1 and the closely related HIV-2 Tat protein. These studies indicated that both the HIV-1 and HIV-2 Tat proteins could specifically interact with RNA polymerase II. Mutagenesis of both HIV-1 and HIV-2 Tat demonstrated that the basic domains of both the HIV-1 and HIV-2 Tat proteins were required for this interaction. Furthermore, "far Western" analysis suggested that the largest subunit of RNA polymerase II was the site for interaction with Tat. The interactions between Tat and RNA polymerase II were of similar magnitude to those detected between RNA polymerase II and the cellular transcription factor RAP30, which stably associates with RNA polymerase II during transcriptional elongation. These studies are consistent with the model that RNA polymerase II is a cellular target for Tat resulting in Tat-mediated increases in transcriptional elongation from the HIV long terminal repeat.

The nuclear matrix protein NMP-1 is the transcription factor YY1.

NMP-1 was initially identified as a nuclear matrix-associated DNA-binding factor that exhibits sequence-specific recognition for the site IV regulatory element of a histone H4 gene. This distal promoter domain is a nuclear matrix interaction site. In the present study, we show that NMP-1 is the multifunctional transcription factor YY1. Gel-shift and Western blot analyses demonstrate that NMP-1 is immunoreactive with YY1 antibody. Furthermore, purified YY1 protein specifically recognizes site IV and reconstitutes the NMP-1 complex. Western blot and gel-shift analyses indicate that YY1 is present within the nuclear matrix. In situ immunofluorescence studies show that a significant fraction of YY1 is localized in the nuclear matrix, principally but not exclusively associated with residual nucleoli. Our results confirm that NMP-1/YY1 is a ubiquitous protein that is present in both human cells and in rat osteosarcoma ROS 17/2.8 cells. The finding that NMP-1 is identical to YY1 suggests that this transcriptional regulator may mediate gene-matrix interactions. Our results are consistent with the concept that the nuclear matrix may functionally compartmentalize the eukaryotic nucleus to support regulation of gene expression.

Oleic acid modulates mRNA expression of liver X receptor (LXR) and its target genes ABCA1 and SREBP1c in human neutrophils

Purpose: Regulation of liver X receptors (LXRs) is essential for cholesterol homeostasis and inflammation. The present study was conducted to determine whether oleic acid (OA) could regulate mRNA expression of LXRα and LXRα-regulated genes and to assess the potential promotion of oxidative stress by OA in neutrophils. Methods: Human neutrophils were treated with OA at different doses and LXR target gene expression, oxidative stress production, lipid efflux and inflammation state were analyzed. Results: We describe that mRNA synthesis of both LXRα and ABCA1 (a reverse cholesterol transporter) was induced by OA in human neutrophils. This fatty acid enhanced the effects of LXR ligands on ABCA1 and LXR expression, but it decreased the mRNA levels of sterol regulatory element-binding protein 1c (a transcription factor that regulates the synthesis of triglycerides). Although OA elicited a slight oxidative stress in the short term (15–30 min) in neutrophils, it is unlikely that this is relevant for the modulation of transcription in our experimental conditions, which involve longer incubation time (i.e., 6 h). Of physiological importance is our finding that OA depresses intracellular lipid levels and that markers of inflammation, such as ERK1/2 and p38 mitogen-activated protein kinase phosphorylation, were decreased by OA treatment. In addition, 200 μM OA reduced the migration of human neutrophils, another marker of the inflammatory state. However, OA did not affect lipid peroxidation induced by pro-oxidant agents. Conclusions: This work presents for the first time evidence that human neutrophils are highly sensitive to OA and provides novel data in support of a protective role of this monounsaturated acid against the activation of neutrophils during inflammation.

Network topology and the evolution of dynamics in an artificial genetic regulatory network model created by whole genome duplication and divergence

Topological measures of large-scale complex networks are applied to a specific artificial regulatory network model created through a whole genome duplication and divergence mechanism. This class of networks share topological features with natural transcriptional regulatory networks. Specifically, these networks display scale-free and small-world topology and possess subgraph distributions similar to those of natural networks. Thus, the topologies inherent in natural networks may be in part due to their method of creation rather than being exclusively shaped by subsequent evolution under selection. The evolvability of the dynamics of these networks is also examined by evolving networks in simulation to obtain three simple types of output dynamics. The networks obtained from this process show a wide variety of topologies and numbers of genes indicating that it is relatively easy to evolve these classes of dynamics in this model. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

Transcriptional network dynamics in macrophage activation

Transcriptional regulatory networks govern cell differentiation and the cellular response to external stimuli. However, mammalian model systems have not yet been accessible for network analysis. Here, we present a genome-wide network analysis of the transcriptional regulation underlying the mouse macrophage response to bacterial lipopolysaccharide (LPS). Key to uncovering the network structure is our combination of time-series cap analysis of gene expression with in silico prediction of transcription factor binding sites. By integrating microarray and qPCR time-series expression data with a promoter analysis, we find dynamic subnetworks that describe how signaling pathways change dynamically during the progress of the macrophage LPS response, thus defining regulatory modules characteristic of the inflammatory response. In particular, our integrative analysis enabled us to suggest novel roles for the transcription factors ATF-3 and NRF-2 during the inflammatory response. We believe that our system approach presented here is applicable to understanding cellular differentiation in higher eukaryotes. (c) 2006 Elsevier Inc. All rights reserved.

Transcriptional regulation os phenylalaline biosynthesis and utilization

Conifer trees divert large quantities of carbon into the biosynthesis of phenylpropanoids, particularly to generate lignin, an important constituent of wood. Since phenylalanine is the precursor for phenylpropanoid biosynthesis, the precise regulation of phenylalanine synthesis and utilization should occur simultaneously. This crucial pathway is finely regulated primarily at the transcriptional level. Transcriptome analyses indicate that the transcription factors (TFs) preferentially expressed during wood formation in plants belong to the MYB and NAC families. Craven-Bartle et al. (2013) have shown in conifers that Myb8 is a candidate regulator of key genes in phenylalanine biosynthesis involved in the supply of the phenylpropane carbon skeleton necessary for lignin biosynthesis. This TF is able to bind AC elements present in the promoter regions of these genes to activate transcription. Constitutive overexpression of Myb8 in white spruce increased secondary-wall thickening and led to ectopic lignin deposition (Bomal et al. 2008). In Arabidopsis, the transcriptional network controlling secondary cell wall involves NAC-domain regulators operating upstream Myb transcription factors. Functional orthologues of members of this network described have been identified in poplar and eucalyptus, but in conifers functional evidence had only been obtained for MYBs. We have identified in the P. pinaster genome 37 genes encoding NAC proteins, which 3 NAC proteins could be potential candidates to be involved in vascular development (Pascual et al. 2015). The understanding of the transcriptional regulatory network associated to phenylpropanoids and lignin biosynthesis in conifers is crucial for future applications in tree improvement and sustainable forest management. This work is supported by the projects BIO2012-33797, BIO2015-69285-R and BIO-474 References: Bomal C, et al. (2008) Involvement of Pinus taeda MYB1 and MYB8 in phenylpropanoid metabolism and secondary cell wall biogenesis: a comparative in planta analysis. J Exp Bot. 59: 3925-3939. Craven-Bartle B, et al. (2013) A Myb transcription factor regulates genes of the phenylalanine pathway in maritime pine. Plant J, 74: 755-766. Pascual MB, et al. (2015) The NAC transcription factor family in maritime pine (Pinus pinaster): molecular regulation of two genes involved in stress responses. BMC Plant Biol, 15: 254.

Molecular markers of obesity and diabetes

Recently it has been shown that the consumption of a diet high in saturated fat is associated with impaired insulin sensitivity and increased incidence of type 2 diabetes. In contrast, diets that are high in monounsaturated fatty acids (MUFAs) or polyunsaturated fatty acids (PUFAs), especially very long chain n-3 fatty acids (FAs), are protective against disease. However, the molecular mechanisms by which saturated FAs induce the insulin resistance and hyperglycaemia associated with metabolic syndrome and type 2 diabetes are not clearly defined. It is possible that saturated FAs may act through alternative mechanisms compared to MUFA and PUFA to regulate of hepatic gene expression and metabolism. It is proposed that, like MUFA and PUFA, saturated FAs regulate the transcription of target genes. To test this hypothesis, hepatic gene expression analysis was undertaken in a human hepatoma cell line, Huh-7, after exposure to the saturated FA, palmitate. These experiments showed that palmitate is an effective regulator of gene expression for a wide variety of genes. A total of 162 genes were differentially expressed in response to palmitate. These changes not only affected the expression of genes related to nutrient transport and metabolism, they also extend to other cellular functions including, cytoskeletal architecture, cell growth, protein synthesis and oxidative stress response. In addition, this thesis has shown that palmitate exposure altered the expression patterns of several genes that have previously been identified in the literature as markers of risk of disease development, including CVD, hypertension, obesity and type 2 diabetes. The altered gene expression patterns associated with an increased risk of disease include apolipoprotein-B100 (apo-B100), apo-CIII, plasminogen activator inhibitor 1, insulin-like growth factor-I and insulin-like growth factor binding protein 3. This thesis reports the first observation that palmitate directly signals in cultured human hepatocytes to regulate expression of genes involved in energy metabolism as well as other important genes. Prolonged exposure to long-chain saturated FAs reduces glucose phosphorylation and glycogen synthesis in the liver. Decreased glucose metabolism leads to elevated rates of lipolysis, resulting in increased release of free FAs. Free FAs have a negative effect on insulin action on the liver, which in turn results in increased gluconeogenesis and systemic dyslipidaemia. It has been postulated that disruption of glucose transport and insulin secretion by prolonged excessive FA availability might be a non-genetic factor that has contributed to the staggering rise in prevalence of type 2 diabetes. As glucokinase (GK) is a key regulatory enzyme of hepatic glucose metabolism, changes in its activity may alter flux through the glycolytic and de novo lipogenic pathways and result in hyperglycaemia and ultimately insulin resistance. This thesis investigated the effects of saturated FA on the promoter activity of the glycolytic enzyme, GK, and various transcription factors that may influence the regulation of GK gene expression. These experiments have shown that the saturated FA, palmitate, is capable of decreasing GK promoter activity. In addition, quantitative real-time PCR has shown that palmitate incubation may also regulate GK gene expression through a known FA sensitive transcription factor, sterol regulatory element binding protein-1c (SREBP-1c), which upregulates GK transcription. To parallel the investigations into the mechanisms of FA molecular signalling, further studies of the effect of FAs on metabolic pathway flux were performed. Although certain FAs reduce SREBP-1c transcription in vitro, it is unclear whether this will result in decreased GK activity in vivo where positive effectors of SREBP-1c such as insulin are also present. Under these conditions, it is uncertain if the inhibitory effects of FAs would be overcome by insulin. The effects of a combination of FAs, insulin and glucose on glucose phosphorylation and metabolism in cultured primary rat hepatocytes at concentrations that mimic those in the portal circulation after a meal was examined. It was found that total GK activity was unaffected by an increased concentration of insulin, but palmitate and eicosapentaenoic acid significantly lowered total GK activity in the presence of insulin. Despite the fact that total GK enzyme activity was reduced in response to FA incubation, GK enzyme translocation from the inactive, nuclear bound, to active, cytoplasmic state was unaffected. Interestingly, none of the FAs tested inhibited glucose phosphorylation or the rate of glycolysis when insulin is present. These results suggest that in the presence of insulin the levels of the active, unbound cytoplasmic GK are sufficient to buffer a slight decrease in GK enzyme activity and decreased promoter activity caused by FA exposure. Although a high fat diet has been associated with impaired hepatic glucose metabolism, there is no evidence from this thesis that FAs themselves directly modulate flux through the glycolytic pathway in isolated primary hepatocytes when insulin is also present. Therefore, although FA affected expression of a wide range of genes, including GK, this did not affect glycolytic flux in the presence of insulin. However, it may be possible that a saturated FA-induced decrease in GK enzyme activity when combined with the onset of insulin resistance may promote the dys-regulation of glucose homeostasis and the subsequent development of hyperglycaemia, metabolic syndrome and type 2 diabetes.

The role of insulin and IGF2 signalling on metabolic pathways in prostate cancer progression

Prostate cancer (CaP) is the most commonly diagnosed cancer in males in Australia, North America, and Europe. If found early and locally confined, CaP can be treated with radical prostatectomy or radiation therapy; however, 25-40% patients will relapse and go on to advanced disease. The most common therapy in these cases is androgen deprivation therapy (ADT), which suppresses androgen production from the testis. Lack of the testicular androgen supply causes cells of the prostate to undergo apoptosis. However, in some cases the regression initially seen with ADT eventually gives way to a growth of a population of cancerous cells that no longer require testicular androgens. This phenotype is essentially fatal and is termed castrate resistant prostate cancer (CRPC). In addition to eventual regression, there are many undesirable side effects which accompany ADT, including development of a metabolic syndrome, which is defined by the U.S. National Library of Medicine as “a combination of medical disorders that increase the risk of developing cardiovascular disease and diabetes.” This project will focus on the effect of ADT induced hyperinsulinemia, as mimicked by treating androgen receptor positive CaP cells with insulin in a serum (hormone) deprived environment. While this side effect is not widely explored, in this thesis it is demonstrated for the first time that insulin upregulates pathways important to CaP progression. Our group has previously shown that during CaP progression, the enzymes necessary for de novo steroidogenesis are upregulated in the LNCaP xenograft model, total steroid levels are increased in tumours compared to pre castrate levels, and de novo steroidogenesis from radio-labelled acetate has been demonstrated. Because of the CaP dependence on AR for survival, we and other groups believe that CaP cells carry out de novo steroidogenesis to survive in androgen deprived conditions. Because (a) men on ADT often develop metabolic syndrome, and (b) men with lifestyle-induced obesity and hyperinsulinemia have worse prognosis and faster disease progression, and because (c) insulin causes steroidogenesis in other cell lines, the hypothesis that insulin may contribute to CaP progression through upregulation of steroidogenesis was explored. Insulin upregulates steroidogenesis enzymes at the mRNA level in three AR positive cell lines, as well as upregulating these enzymes at the protein level in two cell lines. It has also been demonstrated that insulin increases mitochondrial (functional) levels of steroid acute regulatory protein (StAR). Furthermore, insulin causes increased levels of total steroids in and induction of de novo steroid synthesis by insulin has been demonstrated at levels induced sufficient to activate AR. The effect of insulin analogs on CaP steroidogenesis in LNCaP and VCaP cells has also been investigated because epidemiological studies suggest that some of the analogs developed may have more cancer stimulatory effects than normal insulin. In this project, despite the signalling differences between glargine, X10, and insulin, these analogs did not appear to induce steroidogenesis any more potently that normal insulin. The effect of insulin of MCF7breast cancer cells was also investigated with results suggesting that breast cancer cells may be capable of de novo steroidogenesis, and that increase in estradiol production may be exacerbated by insulin. Insulin has also been long known to stimulate lipogenesis in the liver and adipocytes, and has been demonstrated to increase lipogenesis in breast cancer cells; therefore, investigation of the effect of insulin on lipogenesis, which is a hallmark of aggressive cancers, was investigated. In CaP progression sterol regulatory element binding protein (SREBP) is dysregulated and upregulates fatty acid synthase (FASN), acetyl CoA-carboxylase, and other lipogenesis genes. SREBP is important for steroidogenesis and in this project has been shown to be upregulated by insulin in CaP cells. Fatty acid synthesis provides building blocks of membrane growth, provides substrates for acid oxidation, the main energy source for CaP cells, provides building blocks for anti-apoptotic and proinflammatory molecules, and provides molecules that stimulate steroidogenesis. In this project it has been shown that insulin upregulates FASN and ACC, which synthesize fatty acids, as well as upregulating hormone sensitive lipase (HSL), diazepam-binding inhibitor (DBI), and long-chain acyl-CoA synthetase 3 (ACSL3), which contribute to lipid activation of steroidogenesis. Insulin also upregulates total lipid levels and de novo lipogenesis, which can be suppressed by inhibition of the insulin receptor (INSR). The fatty acids synthesized after insulin treatment are those that have been associated with CaP; furthermore, microarray data suggests insulin may upregulate fatty acid biosynthesis, metabolism and arachidonic acid metabolism pathways, which have been implicated in CaP growth and survival. Pharmacological agents used to treat patients with hyperinsulinemia/ hyperlipidemia have gained much interest in regards to CaP risk and treatment; however, the scientific rationale behind these clinical applications has not been examined. This thesis explores whether the use of metformin or simvastatin would decrease either lipogenesis or steroidogenesis or both in CaP cells. Simvastatin is a 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) inhibitor, which blocks synthesis of cholesterol, the building block of steroids/ androgens. It has also been postulated to down regulate SREBP in other metabolic disorders. It has been shown in this thesis, in LNCaP cells, that simvastatin inhibited and decreased insulin induced steroidogenesis and lipogenesis, respectively, but increased these pathways in the absence of insulin. Conversely, metformin, which activates AMP-activated protein kinase (AMPK) to shut down lipogenesis, cholesterol synthesis, and protein synthesis, highly suppresses both steroidogenesis and lipogenesis in the presence and absence of insulin. Lastly, because it has been demonstrated to increase steroidogenesis in other cell lines, and because the elucidation of any factors affecting steroidogenesis is important to understanding CaP, the effect of IGF2 on steroidogenesis in CaP cells was investigated. In patient samples, as men progress to CRPC, IGF2 mRNA and the protein levels of the receptors it may signal through are upregulated. It has also been demonstrated that IGF2 upregulates steroidogenic enzymes at both the mRNA and protein levels in LNCaP cells, increases intracellular and secreted steroid/androgen levels in LNCaPs to levels sufficient to stimulate the AR, and upregulated de novo steroidogenesis in LNCaPs and VCaPs. As well, inhibition of INSR and insulin-like growth factor 1 receptor (IGF1R), which IGF2 signals through, suggests that induction of steroidogenesis may be occurring predominantly through IGF1R. In summary, this project has illuminated for the first time that insulin is likely to play a large role in cancer progression, through upregulation of the steroidogenesis and lipogenesis pathways at the mRNA and protein levels, and production levels, and demonstrates a novel role for IGF-II in CaP progression through stimulation of steroidogenesis. It has also been demonstrated that metformin and simvastatin drugs may be useful in suppressing the insulin induction of these pathways. This project affirms the pathways by which ADT- induced metabolic syndrome may exacerbate CaP progression and strongly suggests that the monitoring and modulation of the metabolic state of CaP patients could have a strong impact on their therapeutic outcomes.

In vitro primary cell culture as a physiologically relevant method for preclinical testing of human oncolytic adenovirus

Ad[I/PPT-E1A] is an oncolytic adenovirus that specifically kills prostate cells via restricted replication by a prostate-specific regulatory element. Off-target replication of oncolytic adenoviruses would have serious clinical consequences. As a proposed ex vivo test, we describe the assessment of the specificity of Ad[I/PPT-E1A] viral cytotoxicity and replication in human nonprostate primary cells. Four primary nonprostate cell types were selected to mimic the effects of potential in vivo exposure to Ad[I/PPT-E1A] virus: bronchial epithelial cells, urothelial cells, vascular endothelial cells, and hepatocytes. Primary cells were analyzed for Ad[I/PPT-E1A] viral cytotoxicity in MTS assays, and viral replication was determined by hexon titer immunostaining assays to quantify viral hexon protein. The results revealed that at an extreme multiplicity of infection of 500, unlikely to be achieved in vivo, Ad[I/PPT-E1A] virus showed no significant cytotoxic effects in the nonprostate primary cell types apart from the hepatocytes. Transmission electron microscopy studies revealed high levels of Ad[I/PPT-E1A] sequestered in the cytoplasm of these cells. Adenoviral green fluorescent protein reporter studies showed no evidence for nuclear localization, suggesting that the cytotoxic effects of Ad[I/PPT-E1A] in human primary hepatocytes are related to viral sequestration. Also, hepatocytes had increased amounts of coxsackie adenovirus receptor surface protein. Active viral replication was only observed in the permissive primary prostate cells and LNCaP prostate cell line, and was not evident in any of the other nonprostate cells types tested, confirming the specificity of Ad[I/PPT-E1A]. Thus, using a relevant panel of primary human cells provides a convenient and alternative preclinical assay for examining the specificity of conditionally replicating oncolytic adenoviruses in vivo.

Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants

Background We describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes. Results We give an example of expression of heme-thiolate P450 to demonstrate effectiveness of this system. We have also designed vectors that take advantage of a dual luciferase assay system to analyse promoter sequences or post-transcriptional regulation of gene expression. We have demonstrated their utility by co-expression of putative transcription factors and the promoter sequence of potential target genes and show how orthologous promoter sequences respond to these genes. Finally, we have constructed a vector that has allowed us to investigate design features of hairpin constructs related to their ability to initiate RNA silencing, and have used these tools to study cis-regulatory effect of intron-containing gene constructs. Conclusion In developing a series of vectors ideally suited to transient expression analysis we have provided a resource that further advances the application of this technology. These minimal vectors are ideally suited to conventional cloning methods and we have used them to demonstrate their flexibility to investigate enzyme activity, transcription regulation and post-transcriptional regulatory processes in transient assays.

Drosophila 3′ UTRs are more complex than protein-coding sequences

The 3′ UTRs of eukaryotic genes participate in a variety of post-transcriptional (and some transcriptional) regulatory interactions. Some of these interactions are well characterised, but an undetermined number remain to be discovered. While some regulatory sequences in 3′ UTRs may be conserved over long evolutionary time scales, others may have only ephemeral functional significance as regulatory profiles respond to changing selective pressures. Here we propose a sensitive segmentation methodology for investigating patterns of composition and conservation in 3′ UTRs based on comparison of closely related species. We describe encodings of pairwise and three-way alignments integrating information about conservation, GC content and transition/transversion ratios and apply the method to three closely related Drosophila species: D. melanogaster, D. simulans and D. yakuba. Incorporating multiple data types greatly increased the number of segment classes identified compared to similar methods based on conservation or GC content alone. We propose that the number of segments and number of types of segment identified by the method can be used as proxies for functional complexity. Our main finding is that the number of segments and segment classes identified in 3′ UTRs is greater than in the same length of protein-coding sequence, suggesting greater functional complexity in 3′ UTRs. There is thus a need for sustained and extensive efforts by bioinformaticians to delineate functional elements in this important genomic fraction. C code, data and results are available upon request.