Methylation analysis in spontaneous sputum for lung cancer diagnosis.

OBJECTIVES: Lung cancer is the most fatal cancer in the developed world due to presence of metastases at time of diagnosis. The aim of this study is to examine DNA hypermethylation in sputum compared to sputum cytology for the diagnosis of lung cancer. A novel risk analysis is introduced, using the distinction between diagnostic and risk markers. METHODS: Two independent sets were randomly composed from a prospectively collected sputum bank (Set 1: n = 98 lung cancer patients, n = 90 controls; Set 2: n = 60 lung cancer patients, n = 445 controls). Sputum cytology was performed for all samples. The following DNA hypermethylation markers were tested in both sets: RASSF1A, APC and cytoglobin (CYGB). Two statistical analyses were conducted: multivariate logistic regression and a risk classification model based on post-test probabilities. RESULTS: In multivariate analysis, RASSF1A was the best of the three markers in discriminating lung cancer cases from controls in both sets (sensitivity 41-52%, specificity 94-96%). The risk model showed that 36% of lung cancer patients were defined as "high risk" (≥ 60% chance on lung cancer) based on RASSF1A hypermethylation in Set 1. The model was reproducible in Set 2. Risk markers (APC, CYGB) have less diagnostic value. Sensitivity of cytology for lung cancer diagnosis was 22%. RASSF1A hypermethylation yielded a sensitivity of 45%. The combined sensitivity for RASSF1A with cytological diagnosis increased to 52% with similar specificity (94%). CONCLUSION: In a diagnostic setting, hypermethylation analysis in sputum is possible when a diagnostic marker is used. However, risk markers are insufficient for this purpose.

Predictions of U.K. regulated power station contributions to regional air pollution and deposition: a model comparison exercise.

Contributions of the emissions from a U.K. regulated fossil-fuel power station to regional air pollution and deposition are estimated using four air quality modeling systems for the year 2003. The modeling systems vary in complexity and emphasis in the way they treat atmospheric and chemical processes, and include the Community Multiscale Air Quality (CMAQ) modeling system in its versions 4.6 and 4.7, a nested modeling system that combines long- and short-range impacts (referred to as TRACK-ADMS [Trajectory Model with Atmospheric Chemical Kinetics-Atmospheric Dispersion Modelling System]), and the Fine Resolution Atmospheric Multi-pollutant Exchange (FRAME) model. An evaluation of the baseline calculations against U.K. monitoring network data is performed. The CMAQ modeling system version 4.6 data set is selected as the reference data set for the model footprint comparison. The annual mean air concentration and total deposition footprints are summarized for each modeling system. The footprints of the power station emissions can account for a significant fraction of the local impacts for some species (e.g., more than 50% for SO2 air concentration and non-sea-salt sulfur deposition close to the source) for 2003. The spatial correlation and the coefficient of variation of the root mean square error (CVRMSE) are calculated between each model footprint and that calculated by the CMAQ modeling system version 4.6. The correlation coefficient quantifies model agreement in terms of spatial patterns, and the CVRMSE measures the magnitude of the difference between model footprints. Possible reasons for the differences between model results are discussed. Finally, implications and recommendations for the regulatory assessment of the impact of major industrial sources using regional air quality modeling systems are discussed in the light of results from this case study.

Circulating DNA is a non-invasive prognostic factor for survival in non-small cell lung cancer.

INTRODUCTION: Circulating plasma DNA is present in a considerably higher concentration in lung cancer patients than in controls. Conflicting data are reported about circulating DNA as a prognostic factor. The aim of this study was to prospectively analyse the relationship of circulating plasma DNA with overall survival (OS) of previously untreated non-small cell lung cancer (NSCLC) patients. METHODS: 46 untreated NSCLC patients and 21 controls with a follow-up time of 6.5 years were analyzed. Quantification of baseline circulating plasma DNA was performed by a real-time quantitative polymerase chain reaction (qPCR) targeting the human beta-globin gene. Survival analysis was performed using the Kaplan-Meier method and compared with a Cox-regression analysis. RESULTS: The median DNA concentration of the patients who died (87%) was significantly higher compared to the patients that survived at the end of follow-up (55ng/ml versus 23ng/ml, p=0.02). In patients with higher DNA concentration overall survival was significantly worse. In this study no relation of DNA concentration with tumour characteristics, age, gender or pulmonary inflammatory conditions was found. CONCLUSION: In this study a high circulating plasma DNA concentration at time of diagnosis in NSCLC patients was a prognostic factor for poorer survival. Circulating DNA may be used as a non-invasive biomarker to refine the prognostic profile in NSCLC patients.

Learning curve of conventional transbronchial needle aspiration in pulmonologists experienced in bronchoscopy.

BACKGROUND: Despite its proven efficacy, transbronchial needle aspiration (TBNA) remains an underutilized technique for sampling enlarged mediastinal lymph nodes in the staging of lung cancer. Previous investigators have reported on TBNA experience, but without mentioning individual learning curves related to lymph node size in pulmonologists experienced in bronchoscopy. OBJECTIVES: The aim of this study was to evaluate the TBNA learning curve in a group of pulmonologists already experienced in bronchoscopy, and to relate their yields to lymph node size and location. METHODS: Data on TBNA yield and related lymph node size were collected retrospectively for five individual pulmonologists. RESULTS: The diagnostic yield of five pulmonologists who started to perform TBNA was evaluated over the first 32 months. TBNA was performed on 138 lymph nodes in 119 patients. The overall diagnostic yield was 77% (range 67-91%). The average diagnostic yield increased from 77% at the start of the learning curve to 82% after 32 months of experience. It was related to lymph node size, but not to lymph node location. The average lymph node size was 22 mm. CONCLUSIONS: Satisfactory results were obtained immediately after introduction of TBNA in the bronchoscopy workup. There is no significant TBNA learning curve. The diagnostic yield was related to lymph node size but not to lymph node location.

Ectonucleotidase activities in Sertoli cells from immature rats.

Sertoli cells have been shown to be targets for extracellular purines such as ATP and adenosine. These purines evoke responses in Sertoli cells through two subtypes of purinoreceptors, P2Y2 and P A1. The signals to purinoreceptors are usually terminated by the action of ectonucleotidases. To demonstrate these enzymatic activities, we cultured rat Sertoli cells for four days and then used them for different assays. ATP, ADP and AMP hydrolysis was estimated by measuring the Pi released using a colorimetric method. Adenosine deaminase activity (EC 3.5.4.4) was determined by HPLC. The cells were not disrupted after 40 min of incubation and the enzymatic activities were considered to be ectocellularly localized. ATP and ADP hydrolysis was markedly increased by the addition of divalent cations to the reaction medium. A competition plot demonstrated that only one enzymatic site is responsible for the hydrolysis of ATP and ADP. This result indicates that the enzyme that acts on the degradation of tri- and diphosphate nucleosides on the surface of Sertoli cells is a true ATP diphosphohydrolase (EC 3.6.1.5) (specific activities of 113 +/- 6 and 21 +/- 2 nmol Pi mg(-1) min(-1) for ATP and ADP, respectively). The ecto-5'-nucleotidase (EC 3.1.3.5) and ectoadenosine deaminase activities (specific activities of 32 +/- 2 nmol Pi mg(-1) min(-1) for AMP and 1.52 +/- 0.13 nmol adenosine mg(-1) min(-1), respectively) were shown to be able to terminate the effects of purines and may be relevant for the physiological control of extracellular levels of nucleotides and nucleosides inside the seminiferous tubules.

Ectonucleotidase activities in Sertoli cells from immature rats

Sertoli cells have been shown to be targets for extracellular purines such as ATP and adenosine. These purines evoke responses in Sertoli cells through two subtypes of purinoreceptors, P2Y2 and P A1. The signals to purinoreceptors are usually terminated by the action of ectonucleotidases. To demonstrate these enzymatic activities, we cultured rat Sertoli cells for four days and then used them for different assays. ATP, ADP and AMP hydrolysis was estimated by measuring the Pi released using a colorimetric method. Adenosine deaminase activity (EC 3.5.4.4) was determined by HPLC. The cells were not disrupted after 40 min of incubation and the enzymatic activities were considered to be ectocellularly localized. ATP and ADP hydrolysis was markedly increased by the addition of divalent cations to the reaction medium. A competition plot demonstrated that only one enzymatic site is responsible for the hydrolysis of ATP and ADP. This result indicates that the enzyme that acts on the degradation of tri- and diphosphate nucleosides on the surface of Sertoli cells is a true ATP diphosphohydrolase (EC 3.6.1.5) (specific activities of 113 + or - 6 and 21 + or - 2 nmol Pi mg-1 min-1 for ATP and ADP, respectively). The ecto-5'-nucleotidase (EC 3.1.3.5) and ectoadenosine deaminase activities (specific activities of 32 + or - 2 nmol Pi mg-1 min-1 for AMP and 1.52 + or - 0.13 nmol adenosine mg-1 min-1, respectively) were shown to be able to terminate the effects of purines and may be relevant for the physiological control of extracellular levels of nucleotides and nucleosides inside the seminiferous tubules

Retinol supplementation induces oxidative stress and modulates antioxidant enzyme activities in rat sertoli cells.

Recent intervention studies revealed that supplementation with retinoids resulted in a higher incidence of lung cancer. Recently the causal mechanism has begun to be clarified. We report here that retinol caused cellular oxidative stress and modulated superoxide dismutase, catalase and glutathione peroxidase activities. Retinol (7 microM) significantly increased TBARS, conjugated dienes, and hydroperoxide-initiated chemiluminescence in cultured Sertoli cells. In response to retinol treatment superoxide dismutase, catalase and glutathione peroxidase activities increased. TBARS content and catalase activities were decreased by a free radical scavenger. These findings suggest that retinol may induce oxidative stress and modulate antioxidant enzyme activities in Sertoli cells.

Mitogenic signaling mediated by oxidants in retinol treated Sertoli cells.

Recent intervention studies revealed that supplementation with retinoids resulted in a higher incidence of lung cancer. Recently the causal mechanism has begun to be clarified. We report here that retinol-induced oxidative stress is accompanied by cellular proliferation. Retinol (7 microM) significantly induced thiobarbituric acid reactive species (TBARS) formation, which was inhibited by trolox, superoxide dismutase, N-acetylcysteine and ethanol. This was accompanied by an increase in DNA synthesis and focus formation in cultured rat Sertoli cells. Antioxidants and ethanol inhibited retinol-induced DNA synthesis. Our findings suggest that retinol-induced oxidative stress was associated with cellular proliferation complementing our understanding of the significance of retinol supplementation in neoplastic transformation.

Retinol-induced elevation of ornithine decarboxylase activity in cultured rat Sertoli cells is attenuated by free radical scavenger and by iron chelator.

We investigated retinol effects in ornithine decarboxylase activity in Sertoli cells. We also tested the hypothesis that free radical scavengers and iron chelators may attenuate the effect of retinol. Sertoli cells isolated from 15-day-old Wistar rats were previously cultured for 48 h and then treated with retinol by 24 h with or without mannitol (1 mM) or 1,10 phenanthroline (100 microM). We measured ornithine decarboxylase and catalase activities and malondialdehyde concentrations in response to retinol treatment. In response to 7 microM retinol treatment ornithine decarboxylase activity increased 30%. Retinol-induced ornithine decarboxylase activity was significantly decreased by addition of free radical scavenger (mannitol) or iron chelator (1,10 phenanthroline). In addition the same effect was observed in catalase increased activity and in malondialdehyde concentrations. These results suggest that retinol treatment induced ornithine decarboxylase and catalase activity and increased malondialdehyde concentration. These effects appear to be mediate by ROS.

Retinol-induced changes in the phosphorylation levels of histones and high mobility group proteins from Sertoli cells.

Chromatin proteins play a role in the organization and functions of DNA. Covalent modifications of nuclear proteins modulate their interactions with DNA sequences and are probably one of the multiple factors involved in the process of switch on/off transcriptionally active regions of DNA. Histones and high mobility group proteins (HMG) are subject to many covalent modifications that may modulate their capacity to bind to DNA. We investigated the changes induced in the phosphorylation pattern of cultured Wistar rat Sertoli cell histones and high mobility group protein subfamilies exposed to 7 microM retinol for up to 48 h. In each experiment, 6 h before the end of the retinol treatment each culture flask received 370 KBq/ml [32P]-phosphate. The histone and HMGs were isolated as previously described [Moreira et al. Medical Science Research (1994) 22: 783-784]. The total protein obtained by either method was quantified and electrophoresed as described by Spiker [Analytical Biochemistry (1980) 108: 263-265]. The gels were stained with Coomassie brilliant blue R-250 and the stained bands were cut and dissolved in 0.5 ml 30% H2O2 at 60oC for 12 h. The vials were chilled and 5.0 ml scintillation liquid was added. The radioactivity in each vial was determined with a liquid scintillation counter. Retinol treatment significantly changed the pattern of each subfamily of histone and high mobility group proteins.

Retinol-induced changes in the phosphorylation levels of histones and high mobility group proteins from Sertoli cells

Chromatin proteins play a role in the organization and functions of DNA. Covalent modifications of nuclear proteins modulate their interactions with DNA sequences and are probably one of the multiple factors involved in the process of switch on/off transcriptionally active regions of DNA. Histones and high mobility group proteins (HMG) are subject to many covalent modifications that may modulate their capacity to bind to DNA. We investigated the changes induced in the phosphorylation pattern of cultured Wistar rat Sertoli cell histones and high mobility group protein subfamilies exposed to 7 µM retinol for up to 48 h. In each experiment, 6 h before the end of the retinol treatment each culture flask received 370 KBq/ml [32P]-phosphate. The histone and HMGs were isolated as previously described [Moreira et al. Medical Science Research (1994) 22: 783-784]. The total protein obtained by either method was quantified and electrophoresed as described by Spiker [Analytical Biochemistry (1980) 108: 263-265]. The gels were stained with Coomassie brilliant blue R-250 and the stained bands were cut and dissolved in 0.5 ml 30 per cent H2O2 at 60oC for 12 h. The vials were chilled and 5.0 ml scintillation liquid was added. The radioactivity in each vial was determined with a liquid scintillation counter. Retinol treatment significantly changed the pattern of each subfamily of histone and high mobility group proteins.

Tamoxifen inhibits particulate-associated protein kinase C activity, and sensitises cultured human glioblastoma cells not to etoposide but to gamma-radiation and BCNU.

We investigated the potential mechanisms of tamoxifen cytotoxicity in the U-373, U-138, and U-87 human glioblastoma cell lines, namely interference with protein kinase C (PKC) activity, the oestrogen receptor, and/or the production of transforming growth factor beta 1 (TGF-beta 1). We further examined the effects of tamoxifen on the cytotoxicity exerted by gamma-radiation, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), and etoposide in this cell line panel. Thus, the cells were treated for 4 days with tamoxifen, gamma-radiation, purified recombinant human TGF-beta 1 (rhTGF-beta 1), BCNU, or etoposide, either alone or at certain combinations. Cellular responses were evaluated with the sulphorhodamine B assay, as well as by multiple drug effect analysis, and related to PKC activities in particulate and cellular fractions; cellular oestrogen receptor contents; and the influence of rhTGF-beta 1 on cell growth. Tamoxifen inhibited cell proliferation as well as the phosphorylation capacity of the particulate, but not of the cytosolic fractions dose-dependently, at comparable kinetics, and at IC50 values of approximately 15 microM. At these concentrations, tamoxifen acted synergistically with gamma-radiation (4- to 6-fold) and additively with BCNU (approximately 2-fold), but did not affect etoposide cytotoxicity. The cells were negative to immunostaining for the oestrogen receptor, and rhRGF-beta 1 did not influence their growth up to 100 nm. Our data suggest that tamoxifen can sensitise cultured glioblastoma cells not to etoposide but to gamma-radiation and BCNU, possibly through interference with membrane PKC, supporting its evaluation in experimental protocols for primary malignant gliomas.

Phenotype modulation of cellular UV-sensitivity.

Mammalian cell shape is critically important to cell differentiation, apoptosis, cell division, and growth arrest. In the present study we examined the relationship among cell density, cell phenotype (which include shape and coupling) and cell survival using the human A549, H596 and H520 non-small cell lung carcinoma lines. Thus, cells from monolayers, aggregated and suspended cultures at different densities were exposed to UV-radiation and both the density and the phenotype of the cells induce shifts in cellular growth rate. Except in suspended cultures, we observed a UV-sensitivity closely related to the proliferative status of the cells. The variability of the cellular response to UV were investigated taking into account the shape and the coupling potential of the cell lines, suggesting that an intercellular-contact mechanism provides further protection against UV-radiation damage.

An Mn2+-stimulated neutral-sphingomyelinase in seminiferous tubules of immature Wistar rats.

Mammalian sphingomyelinases have been implicated in many important physiological and pathophysiological processes. The seminiferous tubules of immature (19 day-old) Wistar rats have at least three types of sphingomyelinases, a lysosomal one and two microsomal ones. One of the microsomal sphingomyelinases is active at pH 6.5 and is stimulated by Mn2+ > Co2+ > Mg2+, and the other is active at pH 7.4 and is stimulated by Mn2+ > Mg2+ and inhibited by Co2+. The two microsomal enzymes are only slightly inhibited by EDTA and at pH 7.4 the stimulatory effects of Mn2+ and Mg2+ are additive. These data characterize the existence of two different membrane-bound sphingomyelinases in the seminiferous tubules of the rat.

GD3 and GM2 synthase activities in rat testes during the period of sexual development.

Activities of two key enzymes of gangliosides biosynthesis were determined in rat testes during development. GD3 synthase activity was low and showed small variations with age. GM2 synthase activity increased 10-fold in testes from 10- to 30-d-old animals, showing a maximum activity at 30 d, followed by a small decrease until 45 d and then a constant activity up to adulthood. These developmental changes in the activity of both glycosyltransferases were related to the increasing complexity in the ganglioside pattern observed in rats testes during the period of sexual development.

Effect of FSH and insulin on lipogenesis in cultures of Sertoli cells from immature rats.

Follicle-stimulating hormone (FSH) and insulin regulate glycide metabolism in Sertoli cells, thus stimulating lactate production. These stimulatory effects of FSH and insulin do not require protein synthesis, suggesting a modulation of enzyme activity and/or regulation of glucose transport. The present investigation was performed to characterize the hormonal control of lipid metabolism in Sertoli cells. The data indicate that FSH and insulin have a regulatory effect on lipid metabolism in Sertoli cells. After 8 h of preincubation with insulin (5 micrograms/ml), the activity of the enzyme ATP-citrate lyase in cultured Sertoli cells was increased from 0.19 to 0.34 nmol NAD+ formed microgram protein-1 min-1. FSH (100 ng/ml) had no effect on this enzyme. Glycerol phosphate dehydrogenase activity was not affected by any of the hormones tested. When Sertoli cells from 19-day old rats were incubated with [1,2-14C]acetate for 90 or 360 min, the [14C] label was present predominantly in triglyceride and phospholipid fractions with minor amounts in other lipids. In Sertoli cells pretreated for 16 h with insulin and FSH, an increase in acetate incorporation into lipids was observed. Most of the label was in esterified lipids and this percentage increased with the time of treatment; this increase was remarkable in triglycerides of control cells (18.8% to 30.6%). Since Sertoli cell triglycerides participate in the control of spermatogenesis, the present data suggest that the hormonal control of lipid metabolism in Sertoli cells is important not only for maintaining the energy of the cell itself, but also for the control of the spermatogenesis process.

Effect of FSH and insulin on lipogenesis in cultures of Sertoli cells from immature rats

Follicle-stimulating hormone (FSH) and insulin regulate glycide metabolism in Sertoli cells, thus stimulating lactate production. These stimulatory effects of FSH and insulin do not require protein synthesis, suggesting a modulation of enzyme activity and/or regulation of glucose transport. The present investigation was performed to characterize the hormonal control of lipid metabolism in Sertoli cells. The data indicate that FSH and insulin have a regulatory effect on lipid metabolism in Sertoli cells. After 8 h of preincubation with insulin (5 mug/ml), the activity of the enzyme ATP-citrate lyase in sultured Sertoli cells was increased from 0.19 to 0.34 nmol NAD+ formed mug protein(-1) min(-1). FSH (100 ng/ml) had no effect on this enzyme. Glycerol phosphate dehydrogenase activity was not affected by any of the hormones tested. When Sertoli cells from 19-day old rats were incubated with [1,2-14C] acetate for 90 or 360 min, the [14C] label was present predominantly in triglyceride and phospholipid fractions with minor amounts in other lipids. In Sertoli cells pretreated for 16 h with insulin and FSH, an increase in acetate incorporation into lipids was observed. Most of the label was in esterified lipids and this percentage increased with the time of treatment; this increase was remarkable in triglycerides of control cells (18.8 percent to 30.6 percent). Since Sertoli cell triglycerides participate in the control of spermatogenesis, the present data suggest that the hormonal control of lipid metabolism in Sertoli cells is important not only for maintaining the energy of the cell itself, but also for the control of the spermatogenesis process.

Influence of the biomatrix on the response of Sertoli cells to FSH.

Sertoli cell preparations isolated from 15-day-old Wistar rats were cultured on two different substrates, i.e., plastic and a biomatrix isolated from seminiferous tubules of rat testis. Sertoli cells cultured on a biomatrix acquired a phenotype and morphology more characteristic of in vivo differentiated cells. In order to determine the influence of a biomatrix on the response of Sertoli cells to FSH, on the 7th day of culture, untreated cells, or cells pretreated for 12 h with FSH (1 microgram/ml), were incubated with [U-14C] leucine or [2-3H] mannose. Cells cultured on the biomatrix showed higher [U-14C] leucine and [2-3H] mannose incorporation into proteins and glycoproteins. FSH increased these activities in cells cultured on both substrates, although its stimulating effect was higher on cells cultured on the biomatrix. These results demonstrate that the biomatrix increases protein and glycoprotein synthesis and secretion, and also influences the response of Sertoli cells to FSH.

Biomatrix effect on Sertoli cells phospholipids.

In order to investigate the influence of biomatrix on Sertoli cell morphology and on the phospholipids content, these cells were isolated from testes of 15-day old Wistar rats and plated onto plastic coated with extracellular matrix extracted from seminiferous tubules, here denoted biomatrix. When the Sertoli cells were cultured on biomatrix they did not form a monolayer until day 7 of culture, while cells plated onto plastic did so 48 h after plating. On day 5 of culture, Sertoli cells were incubated for 48 h with 5 microCi/ml 32P. There was no difference in 32P incorporation into lipids of cells plated onto biomatrix or plastic. However, there was a larger amount of phospholipid phosphate in cells plated onto biomatrix than onto plastic. When the phospholipids were analyzed by bidimensional thin-layer chromatography, no differences were detected in their distribution; however, there was a significant decrease in the percentage of sphingomyelin in cells plated onto biomatrix when compared to plastic. These results showed that the cells cultured on biomatrix change their phospholipids content, but not their distribution. The importance of a small reduction in sphingomyelin content remains to be investigated.

Biomatrix effect on Sertoli cells phospholipids

In order to investigate the influence of biomatrix on Sertoli cell morphology and on the phospholipids content, these cells were isolated from tests of 15-day old Wistar rats and plated ontoplastic coated with extracellular matrix extracted froma seminiferous tubules, here denoted biomatix. When the Sertoli cells were cultured on biomatrix they did not from a monolayer until day 7 of culture, while cells plated onto plastic did so 48 h after plating. On day 5 of culture. Sertoli cells were incubated for 48 h with 5 µCi/ml 32P. There was no difference in 32P incorporation into lipids of cells plated onto biomatrix or plastic. However, there was a larger amount of phospholipid phosphate in cells plated onto biomatrix than onto plastic. When the phospholipds were analyzed by bidimensional thin-layer chromatography, no diferences were detected in their distribution; however, there was a significant decrease in the percentage of sphingomyelin in cells plated onto biomatrix when compared to plastic. These results showed that the cells cultured on biomatrix change their phospholipids content, but not their distribution. The importance of a small reduction in sphingomyelin content remains to be investigated