Regulation of hepatic cytochrome P450 expression in mice with intestinal or systemic infections of citrobacter rodentium.

We reported previously that infection of C3H/HeOuJ (HeOu) mice with the murine intestinal pathogen Citrobacter rodentium caused a selective modulation of hepatic cytochrome P450 (P450) gene expression in the liver that was independent of the Toll-like receptor 4. However, HeOu mice are much more sensitive to the pathogenic effects of C. rodentium infection, and the P450 down-regulation was associated with significant morbidity in the animals. Here, we report that oral infection of C57BL/6 mice with C. rodentium, which produced only mild clinical signs and symptoms, produced very similar effects on hepatic P450 expression in this strain. As in HeOu mice, CYP4A mRNAs and proteins were among the most sensitive to down-regulation, whereas CYP4F18 was induced. CYP2D9 mRNA was also induced 8- to 9-fold in the C57BL/6 mice. The time course of P450 regulation followed that of colonic inflammation and bacterial colonization, peaking at 7 to 10 days after infection and returning to normal at 15 to 24 days as the infection resolved. These changes also correlated with the time course of significant elevations in the serum of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor-alpha, as well as of interferon-gamma and IL-2, with serum levels of IL-6 being markedly higher than those of the other cytokines. Intraperitoneal administration of C. rodentium produced a rapid down-regulation of P450 enzymes that was quantitatively and qualitatively different from that of oral infection, although CYP2D9 was induced in both models, suggesting that the effects of oral infection on the liver are not due to bacterial translocation.

Translational regulation of nuclear gene COX4 expression by mitochondrial content of phosphatidylglycerol and cardiolipin in Saccharomyces cerevisiae.

Previous results indicated that translation of four mitochondrion-encoded genes and one nucleus-encoded gene (COX4) is repressed in mutants (pgs1Delta) of Saccharomyces cerevisiae lacking phosphatidylglycerol and cardiolipin. COX4 translation was studied here using a mitochondrially targeted green fluorescence protein (mtGFP) fused to the COX4 promoter and its 5' and 3' untranslated regions (UTRs). Lack of mtGFP expression independent of carbon source and strain background was established to be at the translational level. The translational defect was not due to deficiency of mitochondrial respiratory function but was rather caused directly by the lack of phosphatidylglycerol and cardiolipin in mitochondrial membranes. Reintroduction of a functional PGS1 gene under control of the ADH1 promoter restored phosphatidylglycerol synthesis and expression of mtGFP. Deletion analysis of the 5' UTR(COX4) revealed the presence of a 50-nucleotide fragment with two stem-loops as a cis-element inhibiting COX4 translation. Binding of a protein factor(s) specifically to this sequence was observed with cytoplasm from pgs1Delta but not PGS1 cells. Using HIS3 and lacZ as reporters, extragenic spontaneous recessive mutations that allowed expression of His3p and beta-galactosidase were isolated, which appeared to be loss-of-function mutations, suggesting that the genes mutated may encode the trans factors that bind to the cis element in pgs1Delta cells.

Energy Signaling in the Regulation of Gene Expression during Stress

Maintenance of homeostasis is pivotal to all forms of life. In the case of plants, homeostasis is constantly threatened by the inability to escape environmental fluctuations, and therefore sensitive mechanisms must have evolved to allow rapid perception of environmental cues and concomitant modification of growth and developmental patterns for adaptation and survival. Re-establishment of homeostasis in response to environmental perturbations requires reprogramming of metabolism and gene expression to shunt energy sources from growth-related biosynthetic processes to defense, acclimation, and, ultimately, adaptation. Failure to mount an initial 'emergency' response may result in nutrient deprivation and irreversible senescence and cell death. Early signaling events largely determine the capacity of plants to orchestrate a successful adaptive response. Early events, on the other hand, are likely to be shared by different conditions through the generation of similar signals and before more specific responses are elaborated. Recent studies lend credence to this hypothesis, underpinning the importance of a shared energy signal in the transcriptional response to various types of stress. Energy deficiency is associated with most environmental perturbations due to their direct or indirect deleterious impact on photosynthesis and/or respiration. Several systems are known to have evolved for monitoring the available resources and triggering metabolic, growth, and developmental decisions accordingly. In doing so, energy-sensing systems regulate gene expression at multiple levels to allow flexibility in the diversity and the kinetics of the stress response.

The regulation of Hox gene expression during animal development

Hox genes encode a family of transcriptional regulators that elicit distinct developmental programmes along the head-to-tail axis of animals. The specific regional functions of individual Hox genes largely reflect their restricted expression patterns, the disruption of which can lead to developmental defects and disease. Here, we examine the spectrum of molecular mechanisms controlling Hox gene expression in model vertebrates and invertebrates and find that a diverse range of mechanisms, including nuclear dynamics, RNA processing, microRNA and translational regulation, all concur to control Hox gene outputs. We propose that this complex multi-tiered regulation might contribute to the robustness of Hox expression during development.

Epigenetic and oncogenic regulation of SLC16A7 (MCT2) results in protein over-expression, impacting on signalling and cellular phenotypes in prostate cancer

Monocarboxylate Transporter 2 (MCT2) is a major pyruvate transporter encoded by the SLC16A7 gene. Recent studies pointed to a consistent overexpression of MCT2 in prostate cancer (PCa) suggesting MCT2 as a putative biomarker and molecular target. Despite the importance of this observation the mechanisms involved in MCT2 regulation are unknown. Through an integrative analysis we have discovered that selective demethylation of an internal SLC16A7/MCT2 promoter is a recurrent event in independent PCa cohorts. This demethylation is associated with expression of isoforms differing only in 5'-UTR translational control motifs, providing one contributing mechanism for MCT2 protein overexpression in PCa. Genes co-expressed with SLC16A7/MCT2 also clustered in oncogenic-related pathways and effectors of these signalling pathways were found to bind at the SLC16A7/MCT2 gene locus. Finally, MCT2 knock-down attenuated the growth of PCa cells. The present study unveils an unexpected epigenetic regulation of SLC16A7/MCT2 isoforms and identifies a link between SLC16A7/MCT2, Androgen Receptor (AR), ETS-related gene (ERG) and other oncogenic pathways in PCa. These results underscore the importance of combining data from epigenetic, transcriptomic and protein level changes to allow more comprehensive insights into the mechanisms underlying protein expression, that in our case provide additional weight to MCT2 as a candidate biomarker and molecular target in PCa.

Expression of glucocorticoid and progesterone nuclear receptor genes in archival breast cancer tissue.

BACKGROUND: Previous studies in our laboratory have shown associations of specific nuclear receptor gene variants with sporadic breast cancer. In order to investigate these findings further, we conducted the present study to determine whether expression levels of the progesterone and glucocorticoid nuclear receptor genes vary in different breast cancer grades. METHODS: RNA was extracted from paraffin-embedded archival breast tumour tissue and converted into cDNA. Sample cDNA underwent PCR using labelled primers to enable quantitation of mRNA expression. Expression data were normalized against the 18S ribosomal gene multiplex and analyzed using analysis of variance. RESULTS: Analysis of variance indicated a variable level of expression of both genes with regard to breast cancer grade (P = 0.00033 for glucocorticoid receptor and P = 0.023 for progesterone receptor). CONCLUSION: Statistical analysis indicated that expression of the progesterone nuclear receptor is elevated in late grade breast cancer tissue.

IL-20 is epigenetically regulated in NSCLC and down regulates the expression of VEGF

Background IL-20 is a pleiotrophic member of the IL-10 family and plays a role in skin biology and the development of haematopoietic cells. Recently, IL-20 has been demonstrated to have potential anti-angiogenic effects in non-small cell lung cancer (NSCLC) by down regulating COX-2. Methods The expression of IL-20 and its cognate receptors (IL-20RA/B and IL-22R1) was examined in a series of resected fresh frozen NSCLC tumours. Additionally, the expression and epigenetic regulation of this family was examined in normal bronchial epithelial and NSCLC cell lines. Furthermore, the effect of IL-20 on VEGF family members was examined. Results The expression of IL-20 and its receptors are frequently dysregulated in NSCLC. IL-20RB mRNA was significantly elevated in NSCLC tumours (p < 0.01). Protein levels of the receptors, IL-20RB and IL-22R1, were significantly increased (p < 0.01) in the tumours of NSCLC patients. IL-20 and its receptors were found to be epigenetically regulated through histone post-translational modifications and DNA CpG residue methylation. In addition, treatment with recombinant IL-20 resulted in decreased expression of the VEGF family members at the mRNA level. Conclusions This family of genes are dysregulated in NSCLC and are subject to epigenetic regulation. Whilst the anti-angiogenic properties of IL-20 require further clarification, targeting this family via epigenetic means may be a viable therapeutic option in lung cancer treatment. © 2011 Elsevier Ltd. All rights reserved.

Epigenetics underpinning the regulation of the CXC (ELR+) chemokines in non-small cell lung cancer

Background: Angiogenesis may play a role in the pathogenesis of Non-Small Cell Lung cancer (NSCLC). The CXC (ELR+) chemokine family are powerful promoters of the angiogenic response. Methods: The expression of the CXC (ELR+) family members (CXCL1-3/GROα-γ, CXCL8/IL-8, CXCR1/2) was examined in a series of resected fresh frozen NSCLC tumours. Additionally, the expression and epigenetic regulation of these chemokines was examined in normal bronchial epithelial and NSCLC cell lines. Results: Overall, expression of the chemokine ligands (CXCL1, 2, 8) and their receptors (CXCR1/2) were down regulated in tumour samples compared with normal, with the exception of CXCL3. CXCL8 and CXCR1/2 were found to be epigenetically regulated by histone post-translational modifications. Recombinant CXCL8 did not stimulate cell growth in either a normal bronchial epithelial or a squamous carcinoma cell line (SKMES-1). However, an increase was observed at 72 hours post treatment in an adenocarcinoma cell line. Conclusions: CXC (ELR+) chemokines are dysregulated in NSCLC. The balance of these chemokines may be critical in the tumour microenvironment and requires further elucidation. It remains to be seen if epigenetic targeting of these pathways is a viable therapeutic option in lung cancer treatment. © 2011 Baird et al.

Matrix metalloproteinases 2 and 9 (gelatinases A and B) expression in malignant mesothelioma and benign pleura

Matrix metalloproteinases (MMPs), in particular the gelatinases (MMP-2 and -9), play a significant role in tumour invasion and angiogenesis. The expression and activities of MMPs have not been characterised in malignant mesothelioma (MM) tumour samples. In a prospective study, gelatinase activity was evaluated in homogenised supernatants of snap frozen MM (n = 35), inflamed pleura (IP, n = 12) and uninflammed pleura (UP, n = 14) tissue specimens by semiquantitative gelatin zymography. Matrix metalloproteinases were correlated with clinicopathological factors and with survival using Kaplan-Meier and Cox proportional hazard models. In MM, pro- and active MMP-2 levels were significantly greater than for MMP-9 (P = 0.006, P<0.001). Active MMP-2 was significantly greater in MM than in UP (P=0.04). MMP-2 activity was equivalent between IP and MM, but both pro- and active MMP-9 activities were greater in IP (P=0.02, P=0.009). While there were trends towards poor survival with increasing total and pro-MMP-2 activity (P=0.08) in univariate analysis, they were both independent poor prognostic factors in multivariate analysis in conjunction with weight loss (pro-MMP-2 P = 0.03, total MMP-2 P = 0.04). Total and pro-MMP-2 also contributed to the Cancer and Leukemia Group B prognostic groups. MMP-9 activities were not prognostic. Matrix metalloproteinases, and in particular MMP-2, the most abundant gelatinase, may play an important role in MM tumour growth and metastasis. Agents that reduce MMP synthesis and/or activity may have a role to play in the management of MM. © 2003 Cancer Research UK.

Regulation of EP receptors in non-small cell lung cancer by epigenetic modifications

Background: Cyclooxygenase (COX)-2 is frequently overexpressed in non-small cell lung cancer (NSCLC) and results in increased levels of prostaglandin E2 (PGE 2), an important signalling molecule implicated in tumourigenesis. PGE 2 exerts its effects through the E prostanoid (EP) receptors (EPs1-4). Methods: The expression and epigenetic regulation of the EPs were evaluated in a series of resected fresh frozen NSCLC tumours and cell lines. Results: EP expression was dysregulated in NSCLC being up and downregulated compared to matched control samples. For EPs1, 3 and 4 no discernible pattern emerged. EP2 mRNA however was frequently downregulated, with low levels being observed in 13/20 samples as compared to upregulation in 5/20 samples examined. In NSCLC cell lines DNA CpG methylation was found to be important for the regulation of EP3 expression, the demethylating agent decitabine upregulating expression. Histone acetylation was also found to be a critical regulator of EP expression, with the histone deacteylase inhibitors trichostatin A, phenylbutyrate and suberoylanilide hydroxamic acid inducing increased expression of EPs2-4. Direct chromatin remodelling was demonstrated at the promoters for EPs2-4. Conclusions: These results indicate that EP expression is variably altered from tumour to tumour in NSCLC. EP2 expression appears to be predominantly downregulated and may have an important role in the pathogenesis of the disease. Epigenetic regulation of the EPs may be central to the precise role COX-2 may play in the evolution of individual tumours. © 2009 Elsevier Ltd. All rights reserved.

An evaluation of tumor oxygenation and gene expression in patients with early stage non-small cell lung cancers

Background: To directly assess tumor oxygenation in resectable non - small cell lung cancers (NSCLC) and to correlate tumor pO2 and the selected gene and protein expression to treatment outcomes. Methods: Twenty patients with resectable NSCLC were enrolled. Intraoperative measurements of normal lung and tumor pO2 were done with the Eppendorf polarographic electrode. All patients had plasma osteopontin measurements by ELISA. Carbonic anhydrase-IX (CA IX) staining of tumor sections was done in the majority of patients (n = 16), as was gene expression profiling (n = 12) using cDNA microarrays. Tumor pO2 was correlated with CA IX staining, osteopontin levels, and treatment outcomes. Results: The median tumor pO2 ranged from 0.7 to 46 mm Hg (median, 16.6) and was lower than normal lung pO2 in all but one patient. Because both variables were affected by the completeness of lung deflation during measurement, we used the ratio of tumor/normal lung (T/L) pO2 as a reflection of tumor oxygenation. The median T/L pO 2 was 0.13. T/L pO2 correlated significantly with plasma osteopontin levels (r = 0.53, P = 0.02) and CA IX expression (P = 0.006). Gene expression profiling showed that high CD44 expression was a predictor for relapse, which was confirmed by tissue staining of CD44 variant 6 protein. Other variables associated with the risk of relapse were T stage (P = 0.02), T/L pO2 (P = 0.04), and osteopontin levels (P = 0.001). Conclusions: Tumor hypoxia exists in resectable NSCLC and is associated with elevated expression of osteopontin and CA IX. Tumor hypoxia and elevated osteopontin levels and CD44 expression correlated with poor prognosis. A larger study is needed to confirm the prognostic significance of these factors. © 2006 American Association for Cancer Research.

The use of artificial MicroRNA technology to control gene expression in Arabidopsis thaliana

In plants, double-stranded RNA (dsRNA) is an effective trigger of RNA silencing, and several classes of endogenous small RNA (sRNA), processed from dsRNA substrates by DICER-like (DCL) endonucleases, are essential in controlling gene expression. One such sRNA class, the microRNAs (miRNAs) control the expression of closely related genes to regulate all aspects of plant development, including the determination of leaf shape, leaf polarity, flowering time, and floral identity. A single miRNA sRNA silencing signal is processed from a long precursor transcript of nonprotein-coding RNA, termed the primary miRNA (pri-miRNA). A region of the pri-miRNA is partially self-complementary allowing the transcript to fold back onto itself to form a stem-loop structure of imperfectly dsRNA. Artificial miRNA (amiRNA) technology uses endogenous pri-miRNAs, in which the miRNA and miRNA*(passenger strand of the miRNA duplex) sequences have been replaced with corresponding amiRNA/ amiRNA*sequences that direct highly efficient RNA silencing of the targeted gene. Here, we describe the rules for amiRNA design, as well as outline the PCR and bacterial cloning procedures involved in the construction of an amiRNA plant expression vector to control target gene expression in Arabidopsis thaliana. © 2014 Springer Science+Business Media New York.

Acquired differential regulation of caspase-8 in cisplatin-resistant non-small-cell lung cancer

Failure to efficiently induce apoptosis contributes to cisplatin resistance in non-small-cell lung cancer (NSCLC). Although BCL-2-associated X protein (BAX) and BCL-2 antagonist killer (BAK) are critical regulators of the mitochondrial apoptosis pathway, their requirement has not been robustly established in relation to cisplatin. Here, we show that cisplatin can efficiently bypass mitochondrial apoptosis block caused by loss of BAX and BAK, via activation of the extrinsic death receptor pathway in some model cell lines. Apoptosis resistance following cisplatin can only be observed when both extrinsic and intrinsic pathways are blocked, consistent with redundancy between mitochondrial and death receptor pathways in cisplatin-induced apoptosis. In H460 NSCLC cells, caspase-8 cleavage was shown to be induced by cisplatin and is dependent on death receptor 4, death receptor 5, Fas-associated protein with death domain, acid sphingomyelinase and ceramide synthesis. In contrast, cisplatin-resistant cells fail to activate caspase-8 via this pathway despite conserving sensitivity to death ligand-driven activation. Accordingly, caspase-8 activation block acquired during cisplatin resistance, can be bypassed by death receptor agonism.

Regulation of ROCK1 via Notch1 during breast cancer cell migration into dense matrices

Background The behaviour of tumour cells depends on factors such as genetics and the tumour microenvironment. The latter plays a crucial role in normal mammary gland development and also in breast cancer initiation and progression. Breast cancer tissues tend to be highly desmoplastic and dense matrix as a pre-existing condition poses one of the highest risk factors for cancer development. However, matrix influence on tumour cell gene expression and behaviour such as cell migration is not fully elucidated. Results We generated high-density (HD) matrices that mimicked tumour collagen content of 20 mg/cm3 that were ~14-fold stiffer than low-density (LD) matrix of 1 mg/cm3. Live-cell imaging showed breast cancer cells utilizing cytoplasmic streaming and cell body contractility for migration within HD matrix. Cell migration was blocked in the presence of both the ROCK inhibitor, Y-27632, and the MMP inhibitor, GM6001, but not by the drugs individually. This suggests roles for ROCK1 and MMP in cell migration are complicated by compensatory mechanisms. ROCK1 expression and protein activity, were significantly upregulated in HD matrix but these were blocked by treatment with a histone deacetylase (HDAC) inhibitor, MS-275. In HD matrix, the inhibition of ROCK1 by MS-275 was indirect and relied upon protein synthesis and Notch1. Inhibition of Notch1 using pooled siRNA or DAPT abrogated the inhibition of ROCK1 by MS-275. Conclusion Increased matrix density elevates ROCK1 activity, which aids in cell migration via cell contractility. The upregulation of ROCK1 is epigenetically regulated in an indirect manner involving the repression of Notch1. This is demonstrated from inhibition of HDACs by MS-275, which caused an upregulation of Notch1 levels leading to blockade of ROCK1 expression.

The expression profiles of the galectin gene family in primary and metastatic papillary thyroid carcinoma with particular emphasis on galectin-1 and galectin-3 expression

INTRODUCTION: Galectin family members have been demonstrated to be abnormally expressed in cancer at the protein and mRNA level. This study investigated the levels of galectin proteins and mRNA expression in a large cohort of patients with papillary thyroid carcinoma and matched lymph node metastases with particular emphasis on galectin-1 and galectin-3. METHODS: mRNA expression of galectin family members (1, 2, 3, 4, 7, 8, 9, 10 and 12) were analysed by real-time polymerase chain reaction in 65 papillary thyroid carcinomas, 30 matched lymph nodes with metastatic papillary thyroid carcinoma and 5 non-cancer thyroid tissues. Galectin-1 and 3 protein expression was determined by immunohistochemistry in these samples. RESULTS: Significant expression differences in all tested galectin family members (1, 2, 3, 4, 7, 8, 9, 10 and 12) were noted for mRNA in papillary thyroid carcinomas, with and without lymph node metastasis. Galectin-1 protein was more strongly expressed than galectin-3 protein in papillary thyroid carcinoma. Galectin-1 protein was found to be overexpressed in 32% of primary papillary thyroid carcinomas. A majority of lymph nodes with metastatic papillary thyroid carcinoma (53%) had significantly increased expression of galectin-1 protein, as did 47% of primaries with metastases. Galectin-1 mRNA levels were decreased in the vast majority (94%) of primary thyroid carcinomas that did not have metastases present. Galectin-3 protein levels were noted to be overexpressed in 15% of primary papillary thyroid carcinomas. In primary papillary thyroid carcinoma with lymph node metastases, 32% had over expression of galectin-3 protein. Overexpression of galectin-3 mRNA was noted in 58% of papillary thyroid carcinomas and 64% of lymph nodes bearing metastatic papillary thyroid carcinoma. Also, primary papillary thyroid carcinoma with lymph node metastases had significantly higher expression of galectin-3 mRNA compared to those without lymph node metastases. CONCLUSION: Galectin family members show altered expression at the mRNA level in papillary thyroid cancers. Overexpression of galectin-1 and 3 proteins were noted in papillary thyroid carcinoma with lymph node metastases. The results presented here demonstrated that galectin-1 and galectin-3 expression have important roles in clinical progression of papillary thyroid carcinoma.