Protective effects of leptin during the suckling period against later obesity may be associated with changes in promoter methylation of the hypothalamic pro-opiomelanocortin gene.

Leptin supplementation of neonatal rats during the suckling period protects against being overweight in adulthood and ameliorates the control of food intake. This was associated with changes in the expression of hypothalamic genes involved in the central action of leptin: pro-opiomelanocortin (Pomc), leptin receptor (Lepr) and suppressor of cytokine signalling (Socs3). The purpose of the present study was to determine the methylation status within the promoter regions of these genes and to assess whether the observed changes in the expression levels of these genes could be explained by changes in their methylation status. Male rats were treated daily with an oral physiological dose of leptin or vehicle during the suckling period. After weaning, animals were fed with a normal-fat or a high-fat (HF) diet until aged 6 months. DNA was extracted from the hypothalamus and methylation within the promoter regions of the gene panel was measured by pyrosequencing. Pomc promoter methylation increased in control animals fed the HF diet but decreased in leptin-treated animals. In addition, there was a weak negative correlation between DNA methylation and POMC mRNA levels (P = 0·075). There were no changes in the methylation status of the CpG sites studied within the promoter regions of Lepr and Socs3 in response to leptin or HF treatments. This is the first demonstration that leptin treatment during lactation may programme methylation of an appetite-related gene in the hypothalamus of animals fed HF diets, with possible implications for gene expression and protection against the development of obesity.

Childhood DNA methylation as a marker of early life rapid weight gain and subsequent overweight.

BACKGROUND: High early postnatal weight gain has been associated with childhood adiposity; however, the mechanism remains unknown. DNA methylation is a hypothesised mechanism linking early life exposures and subsequent disease. However, epigenetic changes associated with high early weight gain have not previously been investigated. Our aim was to investigate the associations between early weight gain, peripheral blood DNA methylation, and subsequent overweight/obese. Data from the UK Avon Longitudinal study of Parents and Children (ALSPAC) cohort were used to estimate associations between early postnatal weight gain and epigenome-wide DNA CpG site methylation (Illumina 450 K Methylation Beadchip) in blood in childhood (n = 125) and late adolescence (n = 96). High weight gain in the first year (a change in weight z-scores > 0.67), both unconditional (rapid weight gain) and conditional on birthweight (rapid thrive), was related to individual CpG site methylation and across regions using the meffil pipeline, with and without adjustment for cell type proportions, and with 5% false discovery rate correction. Variation in methylation at high weight gain-associated CpG sites was then examined with regard to body composition measures in childhood and adolescence. Replication of the differentially methylated CpG sites was sought using whole-blood DNA samples from 104 children from the UK Southampton Women's Survey. RESULTS: Rapid infant weight gain was associated with small (+ 1% change) increases in childhood methylation (age 7) for two distinct CpG sites (cg01379158 (NT5M) and cg11531579 (CHFR)). Childhood methylation at one of these CpGs (cg11531579) was also higher in those who experienced rapid weight gain and were subsequently overweight/obese in adolescence (age 17). Rapid weight gain was not associated with differential DNA methylation in adolescence. Childhood methylation at the cg11531579 site was also suggestively associated with rapid weight gain in the replication cohort. CONCLUSIONS: This study identified associations between rapid weight gain in infancy and small increases in childhood methylation at two CpG sites, one of which was replicated and was also associated with subsequent overweight/obese. It will be important to determine whether loci are markers of early rapid weight gain across different, larger populations. The mechanistic relevance of these differentially methylated sites requires further investigation.

Zinc transporters in the mouse placenta show a coordinated regulatory response to changes in dietary zinc intake.

The aim of the study was to determine if the expression of zinc transporters in the mouse placenta is regulated by dietary zinc, commensurate with regulating the supply of zinc to the fetus. Mice were fed diets differing only in the concentration of zinc (moderately zinc-restricted (ZnR)--15 mg Zn/kg; zinc-adequate (ZnA)--50 mg Zn/kg; zinc-supplemented (ZnS)--150 mg Zn/kg) from the onset of pregnancy until collection of tissue at day 17. Compared with mice fed the other diets, fetal weight was reduced in the ZnR group and total non-embryonic weight gain was reduced in mice fed the ZnS diet. Transcript levels of metallothionein and the zinc transporters ZnT1, ZnT4 and ZIP1 were reduced in the placenta of mice fed both the ZnR and ZnS diets compared with mice fed the ZnA diet. Placental ZnT7 and fetal liver metallothionein transcript levels did not differ significantly between mice fed the three diets and placental ZnT5 was reduced in mice fed the ZnS compared with the ZnA diet but did not differ significantly between the ZnA and ZnR diets. The pattern of mRNA expression in placenta was reflected at the protein level for ZnT1. Levels of ZnT5 protein were also highest in mice fed the ZnA diet. Both ZnT1 and ZnT5 were detected in the human villous syncytiotrophoblast by immunohistochemistry. The data indicate that the expression of zinc transporters in mouse placenta is responsive to dietary zinc supply but this modulation of expression is insufficient to maintain optimum fetal nutrition at even a modest level of dietary zinc restriction.

Tumor-specific expression of cytochrome P450 CYP1B1.

Cytochrome P450 CYP1B1 is a recently cloned dioxin-inducible form of the cytochrome P450 family of xenobiotic metabolizing enzymes. An antibody raised against a peptide specific for CYP1B1 was found to recognize CYP1B1 expressed in human lymphoblastoid cells but not to recognize other forms of cytochrome P450, particularly CYP1A1 and CYP1A2. Using this antibody, the cellular distribution and localization of CYP1B1 were investigated by immunohistochemistry in a range of malignant tumors and corresponding normal tissues. CYP1B1 was found to be expressed at a high frequency in a wide range of human cancers of different histogenetic types, including cancers of the breast, colon, lung, esophagus, skin, lymph node, brain, and testis. There was no detectable immunostaining for CYP1B1 in normal tissues. These results provide the basis for the development of novel methods of cancer diagnosis based on the identification of CYP1B1 in tumor cells and the development of anticancer drugs that are selectively activated in tumors by CYP1B1.

Expression of cell cycle control proteins in primary colorectal tumors does not always predict expression in lymph node metastases.

Analysis of tumor markers focuses on expression in primary tumors with the assumption that this is representative of metastatic tumor, against which treatment is targeted. Few studies have compared the expression of such markers in primary and secondary tumors. In this study, several key genes involved in cell cycle regulation were investigated in colorectal tumors and corresponding lymph node metastases. The cell cycle regulators p53, cyclin D1, p21, p27, retinoblastoma protein (Rb), and proliferating cell nuclear antigen (PCNA) were examined in a series of 42 paired samples of primary colorectal and secondary lymph node tumors by immunohistochemistry. Expression of p53, p27, and Rb was similar in virtually all paired samples (p53, 38 of 42; p27, 39 of 42; Rb, 40 of 42), indicating that the pattern of these proteins in colorectal tumors may be used to predict that in lymph node tumors. It also suggests a lack of direct involvement in the metastatic process. A lower concordance for p21 and cyclin D1 staining was observed between primary and secondary tumors (p21, 19 of 42; cyclin D1, 22 of 42). p21 expression was more often observed in primary colorectal cancers, whereas cyclin D1 expression was more frequently seen in lymph node metastases, in keeping with the contrasting roles of these proteins as a cell cycle inhibitor (p21) and activator (cyclin D1). The PCNA-labeling index was found to vary considerably in a number of cases, thus limiting the ability to predict expression of this protein in lymph node metastases from the primary tumor. In addition, PCNA-labeling indices between paired samples were neither consistently higher nor lower, suggesting that the proliferative capacity of tumor cells is not directly related to their ability to metastasize.

Expression of cytochrome P450 CYP1B1 in breast cancer.

The expression of CYP1B1 has been identified in breast cancer using the reverse transcriptase-polymerase chain reaction and immunoblotting. CYP1B1 mRNA was expressed in the majority of breast tumours and immunoblotting of breast tumours identified a single protein band of molecular weight 60 kDa corresponding to the predicted molecular weight of human CYP1B1. This is the first study to identify CYP1B1 expression in a tumour where it may represent a previously unknown pathway for the metabolism of oestradiol and chemotherapeutic drugs.

Therapeutic opportunities from tumour biology in metastatic colon cancer.

Tumour metastasis is the major cause of morbidity and mortality from colorectal cancer. While improvements in quality of life and patient survival have been made over the past 10 years, the majority of patients with metastatic colorectal cancer will die from their disease. As knowledge of the biology of colon cancer and its invasion/metastasis programme evolve, this presents new therapeutic opportunities for pharmacological and genetic intervention. This review discusses the current approaches to metastatic colorectal cancer therapy, details genomic and biological variance between primary and metastatic tumours, and highlights approaches for harnessing these differences to improve therapy.

Evaluation of the epidermal growth factor receptor (EGFR) in colorectal tumours and lymph node metastases.

Overexpression of the epidermal growth factor receptor (EGFR) often correlates with an aggressive tumour phenotype and poor prognosis. To examine the relevance of EGFR in colorectal cancer, we determined the expression of EGFR protein in 249 colorectal adenocarcinomas and 42 lymph node metastases using immunohistochemistry. Moreover, we investigated a (CA)(n) dinucleotide repeat polymorphism of the EGFR gene in a subset of 114 tumours. High levels of EGFR protein were observed in 123/249 (49.4%) samples. EGFR expression in colorectal carcinomas correlated with differentiation grade (P=0.014). However, there were no associations with Dukes' stage, site, patient age or gender. EGFR protein expression did not influence survival in this colorectal cancer patient cohort (P>or=0.05). Expression was not identical in paired colorectal tumours and lymph node metastases, with only 17/42 (40.5%) samples showing equivalent EGFR levels (P>0.05). The distribution of the (CA)(n) dinucleotide repeat alleles in colorectal adenocarcinomas was not associated with EGFR protein expression (P>0.05). These results indicate that while EGFR overexpression is a common event in colorectal carcinogenesis, it does not influence patient prognosis.

Localization of microsomal epoxide hydrolase in normal and neoplastic human kidney.

Microsomal epoxide hydrolase is a xenobiotic metabolizing enzyme that catalyzes the conversion of toxic and carcinogenic epoxides to less toxic dihydrodiols. The cellular localization and distribution of microsomal epoxide hydrolase were investigated for the first time in normal and neoplastic human kidney. Light microscopic immunohistochemical studies using an alkaline phosphatase-anti-alkaline phosphatase technique showed that in normal kidney there was a wide distribution of epoxide hydrolase immunoreactivity. The main localization of epoxide hydrolase immunoreactivity was to the proximal and distal tubule epithelial cells. Strong epoxide hydrolase immunoreactivity was also identified in epithelium of the collecting ducts. In addition, epoxide hydrolase immunoreactivity was present in vascular endothelial cells, including endothelial cells lining glomerular capillaries. Epoxide hydrolase immunoreactivity was identified in all the renal tumors, and in each tumor immunoreactivity for epoxide hydrolase was localized to tumor cells. Immunoblotting of both normal kidney and tumor microsomes confirmed the presence of a single protein band of molecular weight 49 KD corresponding to the molecular weight of human hepatic microsomal epoxide hydrolase.

Polymorphism in the thymidylate synthase promoter enhancer region in colorectal cancer.

Thymidylate synthase (TS) is an important target for chemotherapy drugs such as 5-fluorouracil and raltitrexed. Over-expression of TS has been linked to chemotherapy resistance. A polymorphic tandem repeat sequence in the 5' untranslated region (5'UTR) of the human TS gene (TSER) has been shown to influence TS expression. The presence of a triple tandem repeat (TSER*3) increases in vitro TS expression compared to a double tandem repeat (TSER*2) and is associated with higher in vivo tumor TS activity. The polymorphism of this promoter enhancer region has not been extensively studied in patients with cancer and may represent a possible mechanism of intrinsic resistance to TS inhibitors. In this study, PCR analysis of genomic DNA from 121 patients with colorectal cancer demonstrated 29% of patients were homozygous for TSER*3, 16% were homozygous for TSER*2 and 55% were heterozygous. In 44/45 microdissected tumors the TS enhancer genotype was identical between paired samples of colorectal tumor and normal tissue. In 24 patients receiving a bolus/infusion 5-fluorouracil (5FU) regimen for metastatic colorectal cancer, 22% of non-responders to chemotherapy were homozygous for TSER*2 compared with 40% of responders. Median survival dropped from 16 months for homozygous TSER*2 to 12 months for homozygous TSER*3. This is consistent with previous studies where higher TS expression was associated with poor response to TS inhibitors. Prospective analysis of the influence of the TS polymorphism on patient outcome is warranted.

Application of the enrichment approach to identify putative markers of response to 5-fluorouracil therapy in advanced colorectal carcinomas.

A wide range of tumor response is seen amongst patients with the same stage of colorectal cancer, even with the use of uniform chemotherapy. The significant economic and personal impact of chemotherapy provides the impetus for the identification of markers of response for use in guiding patient treatment. However, practical constraints prevent evaluation of all putative markers in a definitive manner. In this study, the enrichment approach was evaluated by examining the expression of a panel of putative response markers in selected patient populations with advanced colorectal cancer (i.e., those demonstrating the best and the poorest clinical response to a standardized 5-fluorouracil/folinic acid chemotherapy regimen). Patients showing a good response had a significantly increased survival when compared with poor responders (P=0.0013). Markers were then ranked for clinical importance based on differences in expression between the two groups. This allows for the relatively rapid and inexpensive investigation of multiple markers, using defined patient groups. Bcl-2 overexpression in primary colorectal tumor specimens was found to correlate with clinical response of metastatic deposits to chemotherapy (P=0.044), as did the site of the primary tumor (P=0.011). However, no clear association was observed between response status and the other examined factors (p53, PCNA, TP, MMPs 1, 2 or 9, TIMPs 1 or 2, TS, Dukes' stage at initial diagnosis, histological grade, sex or age). This approach has allowed prioritization of markers of clinical response on which larger, statistically definitive studies will be performed.

Matrix metalloproteinase 13 activity is associated with poor prognosis in colorectal cancer.

AIMS: The matrix metalloproteinases (MMPs) are a family of proteolytic enzymes collectively capable of degrading all extracellular matrix components, in particular fibrillar collagen. The importance of this group of proteins in the processes of tumour invasion and metastasis is now widely acknowledged. MMP-13 (collagenase 3) has a central role in the MMP activation cascade. The purpose of this study was to investigate the presence and activity of MMP-13 in colorectal cancer and relate these to clinicopathological features. METHODS: Immunohistochemistry for MMP-13 was performed on formalin fixed, paraffin wax embedded sections of a large series of colorectal cancers (n = 249), all of which had uniform clinical and pathological information available. Immunoreactivity to MMP-13 was detected with a monoclonal antibody to MMP-13 using a Dako TechMate 500 automated immunostaining system. The presence and cellular localisation of MMP-13 was assessed using a semiquantitative scoring system. Gelatin zymography was used to detect and measure MMP-13 activity. The zymography was performed on a subset of the cases studied by immunohistochemistry using two groups of 10 paired Dukes's C tumours and normal samples, selected by either having "good" or "poor" survival. RESULTS: Immunoreactivity to MMP-13 was identified in 91% of cases and immunoreactivity was localised to the cytoplasm of tumour cells. A high MMP-13 staining score showed a trend towards poorer survival. Tumours had significantly greater MMP-13 activity compared with normal colonic mucosa (p < 0.001). Furthermore, the tumour to normal tissue ratio was significantly higher in the poor survival group (p = 0.02). CONCLUSIONS: These results show that MMP-13 is frequently present and active in colorectal cancer and suggest that the activity of MMP-13 is associated with poorer survival in colorectal cancer.

Expression of matrix metalloproteinases 1, 2, 9 and their tissue inhibitors in stage II non-small cell lung cancer: implications for MMP inhibition therapy.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide, with a very poor survival rate. Therefore there is intense scrutiny to provide a better understanding of the molecular and cellular processes involved in this aggressive disease. The matrix metalloproteinases (MMPs) are a large family of extracellular matrix degrading enzymes believed to play a crucial role in tumor invasion and metastasis. MMP inhibitors are now under development as an adjuvant approach to surgical control of NSCLC. However, there is little data available on MMPs or their tissue inhibitors (TIMPs) in NSCLC. Expression of MMP1, MMP2, MMP9, TIMP1 and TIMP2 was assessed in 44 stage II NSCLC. All proteins were found to be expressed at high levels and significant co-expression was observed. These results suggest that a broad spectrum MMP inhibitor is worthy of evaluation as a therapeutic method of reducing tumor invasion and metastasis in stage II NSCLC.

Modeling the performance of direct-detection Doppler lidar systems including cloud and solar background variability.

Previous modeling of the performance of spaceborne direct-detection Doppler lidar systems assumed extremely idealized atmospheric models. Here we develop a technique for modeling the performance of these systems in a more realistic atmosphere, based on actual airborne lidar observations. The resulting atmospheric model contains cloud and aerosol variability that is absent in other simulations of spaceborne Doppler lidar instruments. To produce a realistic simulation of daytime performance, we include solar radiance values that are based on actual measurements and are allowed to vary as the viewing scene changes. Simulations are performed for two types of direct-detection Doppler lidar system: the double-edge and the multichannel techniques. Both systems were optimized to measure winds from Rayleigh backscatter at 355 nm. Simulations show that the measurement uncertainty during daytime is degraded by only approximately 10-20% compared with nighttime performance, provided that a proper solar filter is included in the instrument design.

Diet induced epigenetic changes and their implications for health.

Dietary exposures can have consequences for health years or decades later and this raises questions about the mechanisms through which such exposures are 'remembered' and how they result in altered disease risk. There is growing evidence that epigenetic mechanisms may mediate the effects of nutrition and may be causal for the development of common complex (or chronic) diseases. Epigenetics encompasses changes to marks on the genome (and associated cellular machinery) that are copied from one cell generation to the next, which may alter gene expression, but which do not involve changes in the primary DNA sequence. These include three distinct, but closely inter-acting, mechanisms including DNA methylation, histone modifications and non-coding microRNAs (miRNA) which, together, are responsible for regulating gene expression not only during cellular differentiation in embryonic and foetal development but also throughout the life-course. This review summarizes the growing evidence that numerous dietary factors, including micronutrients and non-nutrient dietary components such as genistein and polyphenols, can modify epigenetic marks. In some cases, for example, effects of altered dietary supply of methyl donors on DNA methylation, there are plausible explanations for the observed epigenetic changes, but to a large extent, the mechanisms responsible for diet-epigenome-health relationships remain to be discovered. In addition, relatively little is known about which epigenomic marks are most labile in response to dietary exposures. Given the plasticity of epigenetic marks and their responsiveness to dietary factors, there is potential for the development of epigenetic marks as biomarkers of health for use in intervention studies.

Bioinformatic interrogation of expression array data to identify nutritionally regulated genes potentially modulated by DNA methylation.

DNA methylation occurs at CpG dinucleotide sites within the genome and is recognised as one of the mechanisms involved in regulation of gene expression. CpG sites are relatively underrepresented in the mammalian genome, but occur densely in regions called CpG islands (CGIs). CGIs located in the promoters of genes inhibit transcription when methylated by impeding transcription factor binding. Due to the malleable nature of DNA methylation, environmental factors are able to influence promoter CGI methylation patterns and thus influence gene expression. Recent studies have provided evidence that nutrition (and other environmental exposures) can cause altered CGI methylation but, with a few exceptions, the genes influenced by these exposures remain largely unknown. Here we describe a novel bioinformatics approach for the analysis of gene expression microarray data designed to identify regulatory sites within promoters of differentially expressed genes that may be influenced by changes in DNA methylation.

Modeling of direct detection Doppler wind lidar. I. The edge technique.

Analytic models, based on a convolution of a Fabry-Perot etalon transfer function with a Gaussian spectral source, are developed for the shot-noise-limited measurement precision of Doppler wind lidars based on the edge filter technique by use of either molecular or aerosol atmospheric backscatter. The Rayleigh backscatter formulation yields a map of theoretical sensitivity versus etalon parameters, permitting design optimization and showing that the optimal system will have a Doppler measurement uncertainty no better than approximately 2.4 times that of a perfect, lossless receiver. An extension of the models to include the effect of limited etalon aperture leads to a condition for the minimum aperture required to match light collection optics. It is shown that, depending on the choice of operating point, the etalon aperture finesse must be 4-15 to avoid degradation of measurement precision. A convenient, closed-form expression for the measurement precision is obtained for spectrally narrow backscatter and is shown to be useful for backscatter that is spectrally broad as well. The models are extended to include extrinsic noise, such as solar background or the Rayleigh background on an aerosol Doppler lidar. A comparison of the model predictions with experiment has not yet been possible, but a comparison with detailed instrument modeling by McGill and Spinhirne shows satisfactory agreement. The models derived here will be more conveniently implemented than McGill and Spinhirne's and more readily permit physical insights to the optimization and limitations of the double-edge technique.

Modeling of Direct Detection Doppler Wind Lidar. II. The Fringe Imaging Technique.

A simple analytic model is developed for the shot-noise-limited measurement precision of Doppler wind lidars based on the fringe imaging technique by use of either molecular or aerosol atmospheric backscatter. The model leads to etalon design parameters for an instrument optimized for precision. The ultimate measurement precision possible is two to four times the limit for a perfect, lossless receiver. The corresponding result for the double-edge Doppler analyzer was a ratio of 2.5, showing that the two methods are little different in this respect. For aerosol backscatter instruments, the wind speed dynamic range of the fringe imager is substantially greater than that for the edge detector. The etalon aperture needed to meet system etendue requirements is derived and shown to be approximately half that of each of the two etalons required by the double-edge technique. A comparison with more detailed modeling of fringe imaging Doppler-shift analyzers shows good agreement for the Rayleigh model and fair for the aerosol version, confirming the validity of this simpler technique for analyzer design and performance prediction.

Single and tandem Fabry-Perot etalons as solar background filters for lidar.

Atmospheric lidar is difficult in daylight because of sunlight scattered into the receiver field of view. In this research methods for the design and performance analysis of Fabry-Perot etalons as solar background filters are presented. The factor by which the signal to background ratio is enhanced is defined as a measure of the performance of the etalon as a filter. Equations for evaluating this parameter are presented for single-, double-, and triple-etalon filter systems. The role of reflective coupling between etalons is examined and shown to substantially reduce the contributions of the second and third etalons to the filter performance. Attenuators placed between the etalons can improve the filter performance, at modest cost to the signal transmittance. The principal parameter governing the performance of the etalon filters is the etalon defect finesse. Practical limitations on etalon plate smoothness and parallelism cause the defect finesse to be relatively low, especially in the ultraviolet, and this sets upper limits to the capability of tandem etalon filters to suppress the solar background at tolerable cost to the signal.

Fabry-Perot etalon aperture requirements for direct detection Doppler wind lidar from Earth orbit.

The design of Fabry-Perot etalons for direct detection Doppler wind lidar from a satellite is considered for two direct detection methods, fringe imaging (multichannel) and double edge. The area solid-angle product of the etalon for each technique is derived and shown to be inherently larger, for a given etalon aperture, for the fringe imager than for the double-edge Doppler analyzer. Modeling of the Doppler measurement accuracy of a spaceflight direct detection wind lidar shows that a very large optical aperture, 2 m or more, is necessary. Optical throughput matching to a 2-m collector requires, for the fringe-imaging Doppler analyzer, an etalon with 60 mm aperture, whereas the double-edge technique would require two etalons of 200 mm aperture, or a split-aperture etalon of 400 mm working aperture. Because the two direct detection methods have been shown to have practically identical intrinsic sensitivities (measurement accuracies per unit signal), this difference in etalon dimensions may be a significant selection consideration.