Publications by authors named "Simon D Spivack"

34 Publications

First-in-human study of inhaled Azacitidine in patients with advanced non-small cell lung cancer.

Lung Cancer 2021 04 17;154:99-104. Epub 2021 Feb 17.

Department of Oncology, Montefiore Medical Center/Albert Einstein College of Medicine, Bronx, NY10461, USA. Electronic address:

Background: Aerosolized Azacitidine has been shown to inhibit orthotopic lung cancer growth and induce re-expression of methylated tumor suppressor genes in murine models. We hypothesized that inhaled Azacitidine is safe and effective in reversing epigenetic changes in the bronchial epithelium secondary to chronic smoking.

Patients And Methods: We report the first in human study of inhaled Azacitidine. Azacitidine in aqueous solution was used to generate an aerosol suspension of 0.25-5 μm particle size. Main inclusion criteria: Stage IV or recurrent NSCLC with predominantly lung involvement, ≥1 prior systemic therapy, ECOG PS 0-1, and adequate pulmonary function. Patients received inhaled Azacitidine daily on days 1-5 and 15-19 of 28-day cycles, at 3 escalating doses (15, 30 and 45 mg/m daily). The primary objective was to determine the feasibility and tolerability of this new therapeutic modality. The key secondary objectives included pharmacokinetics, methylation profiles and efficacy.

Results: From 3/2015 to 2/2018, eight patients received a median number of 2 (IQR = 1) cycles of inhaled Azacitidine. No clinically significant adverse events were observed, except one patient treated at the highest dose developed an asymptomatic grade 2 decreased DLCO which resolved spontaneously. One patient receiving 12 cycles of therapy had an objective and durable partial response, and two patients had stable disease. Plasma Azacitidine was only briefly detectable in patients treated at the higher doses. Moreover, in 2 of 3 participants who agreed and underwent pre- and post-treatment bronchoscopy, the global DNA methylation in the bronchial epithelium decreased by 24 % and 79 % post-therapy, respectively. The interval between last inhaled treatment and bronchoscopy was 3 days.

Conclusions: Inhaled Azacitidine resulted in negligible plasma levels compared to the previously reported subcutaneous administration and was well-tolerated. The results justify the continued development of inhaled Azacitidine at non-cytotoxic doses for patients with lung-confined malignant and/or premalignant lesions.
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http://dx.doi.org/10.1016/j.lungcan.2021.02.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8026712PMC
April 2021

Genetic Variants Associated with FDNY WTC-Related Sarcoidosis.

Int J Environ Res Public Health 2019 05 23;16(10). Epub 2019 May 23.

Pulmonology Division, Department of Medicine, Montefiore Medical Center, Bronx, NY 10467, USA.

Sarcoidosis is a systemic granulomatous disease of unknown etiology. It may develop in response to an exposure or inflammatory trigger in the background of a genetically primed abnormal immune response. Thus, genetic studies are potentially important to our understanding of the pathogenesis of sarcoidosis. We developed a case-control study which explored the genetic variations between firefighters in the Fire Department of the City of New York (FDNY) with World Trade Center (WTC)-related sarcoidosis and those with WTC exposure, but without sarcoidosis. The loci of fifty-one candidate genes related to granuloma formation, inflammation, immune response, and/or sarcoidosis were sequenced at high density in enhancer/promoter, exonic, and 5' untranslated regions. Seventeen allele variants of human leukocyte antigen (HLA) and non-HLA genes were found to be associated with sarcoidosis, and all were within chromosomes 1 and 6. Our results also suggest an association between extrathoracic involvement and allele variants of HLA and non-HLA genes found not only on chromosomes 1 and 6, but also on chromosomes 16 and 17. We found similarities between genetic variants with WTC-related sarcoidosis and those reported previously in sporadic sarcoidosis cases within the general population. In addition, we identified several allele variants never previously reported in association with sarcoidosis. If confirmed in larger studies with known environmental exposures, these novel findings may provide insight into the gene-environment interactions key to the development of sarcoidosis.
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http://dx.doi.org/10.3390/ijerph16101830DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6572061PMC
May 2019

Global, integrated analysis of methylomes and transcriptomes from laser capture microdissected bronchial and alveolar cells in human lung.

Epigenetics 2018 10;13(3):264-274. Epub 2018 May 10.

a Department of Genetics , Albert Einstein College of Medicine , 1301 Morris Park Ave, Bronx , New York 10461 , USA.

Gene regulatory analysis of highly diverse human tissues in vivo is essentially constrained by the challenge of performing genome-wide, integrated epigenetic and transcriptomic analysis in small selected groups of specific cell types. Here we performed genome-wide bisulfite sequencing and RNA-seq from the same small groups of bronchial and alveolar cells isolated by laser capture microdissection from flash-frozen lung tissue of 12 donors and their peripheral blood T cells. Methylation and transcriptome patterns differed between alveolar and bronchial cells, while each of these epithelia showed more differences from mesodermally-derived T cells. Differentially methylated regions (DMRs) between alveolar and bronchial cells tended to locate at regulatory regions affecting promoters of 4,350 genes. A large number of pathways enriched for these DMRs including GTPase signal transduction, cell death, and skeletal muscle. Similar patterns of transcriptome differences were observed: 4,108 differentially expressed genes (DEGs) enriched in GTPase signal transduction, inflammation, cilium assembly, and others. Prioritizing using DMR-DEG regulatory network, we highlighted genes, e.g., ETS1, PPARG, and RXRG, at prominent alveolar vs. bronchial cell discriminant nodes. Our results show that multi-omic analysis of small, highly specific cells is feasible and yields unique physiologic loci distinguishing human lung cell types in situ.
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http://dx.doi.org/10.1080/15592294.2018.1441650DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5997142PMC
February 2019

Clinical Course of Sarcoidosis in World Trade Center-Exposed Firefighters.

Chest 2018 01 21;153(1):114-123. Epub 2017 Oct 21.

Bureau of Health Services, Fire Department of the City of New York, Brooklyn, NY; Pulmonary Division, Department of Medicine, Montefiore Medical Center and the Albert Einstein College of Medicine, Bronx, NY. Electronic address:

Background: Sarcoidosis is believed to represent a genetically primed, abnormal immune response to an antigen exposure or inflammatory trigger, with both genetic and environmental factors playing a role in disease onset and phenotypic expression. In a population of firefighters with post-World Trade Center (WTC) 9/11/2001 (9/11) sarcoidosis, we have a unique opportunity to describe the clinical course of incident sarcoidosis during the 15 years postexposure and, on average, 8 years following diagnosis.

Methods: Among the WTC-exposed cohort, 74 firefighters with post-9/11 sarcoidosis were identified through medical records review. A total of 59 were enrolled in follow-up studies. For each participant, the World Association of Sarcoidosis and Other Granulomatous Diseases organ assessment tool was used to categorize the sarcoidosis involvement of each organ system at time of diagnosis and at follow-up.

Results: The incidence of sarcoidosis post-9/11 was 25 per 100,000. Radiographic resolution of intrathoracic involvement occurred in 24 (45%) subjects. Lung function for nearly all subjects was within normal limits. Extrathoracic involvement increased, most prominently joints (15%) and cardiac (16%) involvement. There was no evidence of calcium dysmetabolism. Few subjects had ocular (5%) or skin (2%) involvement, and none had beryllium sensitization. Most (76%) subjects did not receive any treatment.

Conclusions: Extrathoracic disease was more prevalent in WTC-related sarcoidosis than reported for patients with sarcoidosis without WTC exposure or for other exposure-related granulomatous diseases (beryllium disease and hypersensitivity pneumonitis). Cardiac involvement would have been missed if evaluation stopped after ECG, 48-h recordings, and echocardiogram. Our results also support the need for advanced cardiac screening in asymptomatic patients with strenuous, stressful, public safety occupations, given the potential fatality of a missed diagnosis.
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http://dx.doi.org/10.1016/j.chest.2017.10.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6026251PMC
January 2018

Utility of low-dose oral aspirin challenges for diagnosis of aspirin-exacerbated respiratory disease.

Ann Allergy Asthma Immunol 2016 Apr 25;116(4):321-328.e1. Epub 2016 Jan 25.

Albert Einstein College of Medicine/Montefiore Medical Center, Bronx, New York.

Background: Aspirin-exacerbated respiratory disease (AERD) is diagnosed through graded aspirin challenges that induce hypersensitivity reactions and eicosanoid level changes. It is not known whether diagnostically useful changes also occur after low-dose aspirin challenges that do not induce hypersensitivity reactions.

Objective: To investigate the utility of low-dose oral aspirin challenges for diagnosing AERD by measuring different clinical parameters and eicosanoid changes.

Methods: Sixteen patients with AERD and 13 patients with aspirin-tolerant asthma underwent oral challenges with low-dose (20 or 40 mg) aspirin and diagnostic oral graded aspirin challenges (up to 325 mg of aspirin). Forced expiratory volume in 1 second, nasal peak flow, the fraction of exhaled nitric oxide (FeNO), and eicosanoid levels in plasma and urine were analyzed.

Results: In patients with AERD but not in those with aspirin-tolerant asthma, 40-mg aspirin challenges induced a significant mean (SEM) decrease from baseline in FeNO (19% [5.1%]; P = .001) without causing any hypersensitivity reaction. The FeNO decrease also occurred after higher-dose aspirin challenges (27.8% [4.9%]; P < .001). The sensitivity and specificity of 40-mg aspirin-induced FeNO changes for identifying AERD were 90% and 100% with an area under the curve of 0.98 (95% CI, 0.92-1.00). The low-dose challenge also induced a significant leukotriene E4 urine increase in patients with AERD (from 6.32 [0.08] to 6.91 [0.15] log-pg/mg creatinine; P < .001), but the sensitivity and specificity of these changes were less than for the FeNO changes.

Conclusion: The low-dose aspirin-induced decrease in FeNO in patients with AERD may be useful for the diagnosis of aspirin allergy without inducing a hypersensitivity reaction.

Trial Registration: clinicaltrials.gov Identifier: NCT01320072.
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http://dx.doi.org/10.1016/j.anai.2015.12.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4826295PMC
April 2016

Genome Wide Methylome Alterations in Lung Cancer.

PLoS One 2015 18;10(12):e0143826. Epub 2015 Dec 18.

Department of Medicine/Pulmonary, Albert Einstein College of Medicine, Bronx, New York, United States of America.

Aberrant cytosine 5-methylation underlies many deregulated elements of cancer. Among paired non-small cell lung cancers (NSCLC), we sought to profile DNA 5-methyl-cytosine features which may underlie genome-wide deregulation. In one of the more dense interrogations of the methylome, we sampled 1.2 million CpG sites from twenty-four NSCLC tumor (T)-non-tumor (NT) pairs using a methylation-sensitive restriction enzyme- based HELP-microarray assay. We found 225,350 differentially methylated (DM) sites in adenocarcinomas versus adjacent non-tumor tissue that vary in frequency across genomic compartment, particularly notable in gene bodies (GB; p<2.2E-16). Further, when DM was coupled to differential transcriptome (DE) in the same samples, 37,056 differential loci in adenocarcinoma emerged. Approximately 90% of the DM-DE relationships were non-canonical; for example, promoter DM associated with DE in the same direction. Of the canonical changes noted, promoter (PR) DM loci with reciprocal changes in expression in adenocarcinomas included HBEGF, AGER, PTPRM, DPT, CST1, MELK; DM GB loci with concordant changes in expression included FOXM1, FERMT1, SLC7A5, and FAP genes. IPA analyses showed adenocarcinoma-specific promoter DMxDE overlay identified familiar lung cancer nodes [tP53, Akt] as well as less familiar nodes [HBEGF, NQO1, GRK5, VWF, HPGD, CDH5, CTNNAL1, PTPN13, DACH1, SMAD6, LAMA3, AR]. The unique findings from this study include the discovery of numerous candidate The unique findings from this study include the discovery of numerous candidate methylation sites in both PR and GB regions not previously identified in NSCLC, and many non-canonical relationships to gene expression. These DNA methylation features could potentially be developed as risk or diagnostic biomarkers, or as candidate targets for newer methylation locus-targeted preventive or therapeutic agents.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143826PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684329PMC
June 2016

Lung cancer transcriptomes refined with laser capture microdissection.

Am J Pathol 2014 Nov 14;184(11):2868-84. Epub 2014 Aug 14.

Division of Pulmonary Medicine, Department of Medicine, Albert Einstein College of Medicine, Bronx, New York. Electronic address:

We evaluated the importance of tumor cell selection for generating gene signatures in non-small cell lung cancer. Tumor and nontumor tissue from macroscopically dissected (Macro) surgical specimens (31 pairs from 32 subjects) was homogenized, extracted, amplified, and hybridized to microarrays. Adjacent scout sections were histologically mapped; sets of approximately 1000 tumor cells and nontumor cells (alveolar or bronchial) were procured by laser capture microdissection (LCM). Within histological strata, LCM and Macro specimens exhibited approximately 67% to 80% nonoverlap in differentially expressed (DE) genes. In a representative subset, LCM uniquely identified 300 DE genes in tumor versus nontumor specimens, largely attributable to cell selection; 382 DE genes were common to Macro, Macro with preamplification, and LCM platforms. RT-qPCR validation in a 33-gene subset was confirmatory (ρ = 0.789 to 0.964, P = 0.0013 to 0.0028). Pathway analysis of LCM data suggested alterations in known cancer pathways (cell growth, death, movement, cycle, and signaling components), among others (eg, immune, inflammatory). A unique nine-gene LCM signature had higher tumor-nontumor discriminatory accuracy (100%) than the corresponding Macro signature (87%). Comparison with Cancer Genome Atlas data sets (based on homogenized Macro tissue) revealed both substantial overlap and important differences from LCM specimen results. Thus, cell selection via LCM enhances expression profiling precision, and confirms both known and under-appreciated lung cancer genes and pathways.
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http://dx.doi.org/10.1016/j.ajpath.2014.06.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4215031PMC
November 2014

Site-specific methylated reporter constructs for functional analysis of DNA methylation.

Epigenetics 2013 Nov 4;8(11):1176-87. Epub 2013 Sep 4.

Pulmonary Medicine; Albert Einstein College of Medicine; Bronx, NY USA; Genetics; Albert Einstein College of Medicine; Bronx, NY USA.

Methods to experimentally alter and functionally evaluate cytosine methylation in a site-specific manner have proven elusive. We describe a site-specific DNA methylation method, using synthetically methylated primers and high fidelity PCR coupled with ligation of reporter constructs. We applied this method to introduce methylated cytosines into fragments of the respective DAPK and RASSF1A promoters that had been cloned into luciferase reporters. We found that methylation of 3-7 residue CpG clusters that were 5' adjacent to the transcription start site (TSS) of the DAPK gene produced up to a 54% decrease in promoter activity (p<0.01). Similarly, for RASSF1A promoter reporter constructs, the methylation of either of two clusters of four CpGs each, but not an intervening cluster, produced a 63% decrease in promoter activity (p<0.01), suggesting that precise mCpG position is crucial, and factors other than simple proximity to the TSS are at play. Chromatin immunoprecipitation analysis of these reporter constructs demonstrated that transcription factor Oct-1 and Sp1 preferentially bound the unmethylated vs. methylated DAPK or RASSF1A promoter reporter constructs at the functional CpG sites. Histone H1, hnRNP1, and MeCP2 showed preferential binding to methylated sequence at functional sites in these reporter constructs, as well as highly preferential (> 8-80-fold) binding to native methylated vs. unmethylated chromatin. These results suggest that: (1) site-specific, precision DNA methylation of a reporter construct can be used for functional analysis of commonly observed gene promoter methylation patterns; (2) the reporter system contains key elements of the endogenous chromatin machinery.
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http://dx.doi.org/10.4161/epi.26195DOI Listing
November 2013

A quantitative method to identify microRNAs targeting a messenger RNA using a 3'UTR RNA affinity technique.

Anal Biochem 2013 Dec 9;443(1):1-12. Epub 2013 Aug 9.

Division of Pulmonary Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

The identification of specific microRNAs (miRNAs) that target a given messenger RNA (mRNA) is essential for studies in gene regulation, but the available bioinformatic software programs are often unreliable. We have developed a unique experimental miRNA affinity assay whereby a 3'UTR RNA is end-labeled with biotin, immobilized, and then used as a bait sequence for affinity pull-down of miRNAs. After washes and release, cloning and sequencing identify the miRNAs. Binding affinity is quantitated by quantitative polymerase chain reaction (qPCR), comparing released and original input concentrations. As an initial demonstration, the TCF8/ZEB1 mRNA affinity pull-down yielded miR-200 family member miRs in the majority of clones, and binding affinity was approximately 100%; virtually all copies of miR-200c bound the immobilized mRNA transcript. For validation in cells, miR-200c strongly inhibited expression of a TCF8 luciferase reporter, native TCF8 mRNA, and protein levels, which contrasted with other recovered miRNAs with lower binding affinities. For Smad4 mRNA, miR-150 (and others) displayed a binding affinity of 39% (or less) yet did not inhibit a Smad4 reporter, native Smad4 mRNA, or protein levels. These results were not predicted by available software. This work demonstrates this miRNA binding affinity assay to be a novel yet facile experimental means of identification of miRNAs targeting a given mRNA.
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http://dx.doi.org/10.1016/j.ab.2013.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4112567PMC
December 2013

Epidemiology of lung cancer: Diagnosis and management of lung cancer, 3rd ed: American College of Chest Physicians evidence-based clinical practice guidelines.

Chest 2013 May;143(5 Suppl):e1S-e29S

Division of Pulmonary Medicine, Department of Medicine, Albert Einstein College of Medicine, Bronx, NY.

Background: Ever since a lung cancer epidemic emerged in the mid-1900 s, the epidemiology of lung cancer has been intensively investigated to characterize its causes and patterns of occurrence. This report summarizes the key findings of this research.

Methods: A detailed literature search provided the basis for a narrative review, identifying and summarizing key reports on population patterns and factors that affect lung cancer risk.

Results: Established environmental risk factors for lung cancer include smoking cigarettes and other tobacco products and exposure to secondhand tobacco smoke, occupational lung carcinogens, radiation, and indoor and outdoor air pollution. Cigarette smoking is the predominant cause of lung cancer and the leading worldwide cause of cancer death. Smoking prevalence in developing nations has increased, starting new lung cancer epidemics in these nations. A positive family history and acquired lung disease are examples of host factors that are clinically useful risk indicators. Risk prediction models based on lung cancer risk factors have been developed, but further refinement is needed to provide clinically useful risk stratification. Promising biomarkers of lung cancer risk and early detection have been identified, but none are ready for broad clinical application.

Conclusions: Almost all lung cancer deaths are caused by cigarette smoking, underscoring the need for ongoing efforts at tobacco control throughout the world. Further research is needed into the reasons underlying lung cancer disparities, the causes of lung cancer in never smokers, the potential role of HIV in lung carcinogenesis, and the development of biomarkers.
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http://dx.doi.org/10.1378/chest.12-2345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4694610PMC
May 2013

Genetic and epigenetic regulation of AHR gene expression in MCF-7 breast cancer cells: role of the proximal promoter GC-rich region.

Biochem Pharmacol 2012 Sep 21;84(5):722-35. Epub 2012 Jun 21.

Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, contributes to carcinogenesis through its role in the regulation of cytochrome P450 1 (CYP1)-catalyzed metabolism of carcinogens. Here, we investigated genetic and epigenetic mechanisms that affect AhR expression. Analyses of the human AHR proximal promoter in MCF-7 human breast cancer cells using luciferase assays and electrophoretic mobility shift assays revealed multiple specificity protein (Sp) 1 binding sequences that are transcriptional activators in vitro. The regulation of AhR expression was evaluated in long-term estrogen exposed (LTEE) MCF-7 cells, which showed increased AhR expression, enhanced CYP1 inducibility, and increased capacity to form DNA adducts when exposed to the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. The increased AhR expression in LTEE cells was found not to result from increased mRNA stability, differential RNA processing, or decreased DNA methylation. Analysis of the AHR proximal promoter region using chromatin immunoprecipitation confirmed that enhanced expression of AhR in LTEE cells involves changes in histone modifications, notably decreased trimethylation of histone 3, lysine 27. Upon further examination of the GC-rich Sp1-binding region, we confirmed that it contains a polymorphic (GGGGC)(n) repeat. In a population of newborns from New York State, the allele frequency of (GGGGC)(n) was n = 4 > 5 ≫ 6, 2. Circular dichroism spectroscopy revealed the ability of sequences of this GC-rich region to form guanine-quadruplex structures in vitro. These studies revealed multiple levels at which AhR expression may be controlled, and offer additional insights into mechanisms regulating AhR expression that can ultimately impact carcinogenesis.
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http://dx.doi.org/10.1016/j.bcp.2012.06.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3965201PMC
September 2012

High-throughput library screening identifies two novel NQO1 inducers in human lung cells.

Am J Respir Cell Mol Biol 2012 Mar 20;46(3):365-71. Epub 2011 Oct 20.

M.D. Department of Health Science Research, Mayo Clinic, 200 First Street S.W., Rochester, MN 55905, USA.

Many phytochemicals possess antioxidant and cancer-preventive properties, some putatively through antioxidant response element-mediated phase II metabolism, entailing mutagen/oxidant quenching. In our recent studies, however, most candidate phytochemical agents were not potent in inducing phase II genes in normal human lung cells. In this study, we applied a messenger RNA (mRNA)-specific gene expression-based high throughput in vitro screening approach to discover new, potent plant-derived phase II inducing chemopreventive agents. Primary normal human bronchial epithelial (NHBE) cells and immortalized human bronchial epithelial cells (HBECs) were exposed to 800 individual compounds in the MicroSource Natural Products Library. At a level achievable in humans by diet (1.0 μM), 2,3-dihydroxy-4-methoxy-4'-ethoxybenzophenone (DMEBP), triacetylresveratrol (TRES), ivermectin, sanguinarine sulfate, and daunorubicin induced reduced nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase 1 (NQO1) mRNA and protein expression in NHBE cells. DMEBP and TRES were the most attractive agents as coupling potency and low toxicity for induction of NQO1 (mRNA level, ≥3- to 10.8-fold that of control; protein level, ≥ two- to fourfold that of control). Induction of glutathione S-transferase pi mRNA expression was modest, and none was apparent for glutathione S-transferase pi protein expression. Measurements of reactive oxygen species and glutathione/oxidized glutathione ratio showed an antioxidant effect for DMEBP, but no definite effect was found for TRES in NHBE cells. Exposure of NHBE cells to H(2)O(2) induced nuclear translocation of nuclear factor erythroid 2-related factor 2, but this translocation was not significantly inhibited by TRES and DMEBP. These studies show that potency and low toxicity may align for two potential NQO1-inducing agents, DMEBP and TRES.
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http://dx.doi.org/10.1165/rcmb.2011-0301OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326428PMC
March 2012

Lung cancer and its association with chronic obstructive pulmonary disease: update on nexus of epigenetics.

Curr Opin Pulm Med 2011 Jul;17(4):279-85

Department of Environmental Medicine, Lung Biology and Disease Program, University of Rochester Medical Center, Rochester, NY, USA.

Purpose Of Review: Chronic obstructive pulmonary disease (COPD) and lung cancer are the leading causes of morbidity and mortality worldwide. The current research is focused on identifying the common and disparate events involved in epigenetic modifications that concurrently occur during the pathogenesis of COPD and lung cancer. The purpose of this review is to describe the current knowledge and understanding of epigenetic modifications in pathogenesis of COPD and lung cancer.

Recent Findings: This review provides an update on advances of how epigenetic modifications are linked to COPD and lung cancer, and their commonalities and disparities. The key epigenetic modification enzymes (e.g. DNA methyltransferases -- CpG methylation, histone acetylases/deacetylases and histone methyltransferases/demethylases) that are identified to play an important role in COPD and lung tumorigenesis and progression are described in this review.

Summary: Distinct DNA methyltransferases and histone modification enzymes are differentially involved in pathogenesis of lung cancer and COPD, although some of the modifications are common. Understanding the epigenetic modifications involved in pathogenesis of lung cancer or COPD with respect to common and disparate mechanisms will lead to targeting of epigenetic therapies against these disorders.
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http://dx.doi.org/10.1097/MCP.0b013e3283477533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3730439PMC
July 2011

The 3 prime paradigm of the miR-200 family and other microRNAs.

Epigenetics 2011 Mar 1;6(3):268-72. Epub 2011 Mar 1.

Pfizer, Division of Molecular Medicine, Groton, CT, USA.

The number of predicted human microRNAs in Sanger miRBase currently stands at over a thousand, with each of these in turn predicted to target numerous mRNAs. However, those microRNAs for which mRNA targets have been evaluated, verified and reported in the literature are still in the minority and the bulk of microRNA/mRNA interactions are yet to be confirmed. Confirmation of microRNA interaction with predicted mRNA targets represents a considerable undertaking, made more complex by potential synergistic effects of multiple microRNAs and the three possible outcomes (translational repression, degradation or a mixture of both). In addition, contrasting results obtained when either stably expressing or transiently transfecting members of the miR-200 family illustrate limitations in the verification methods currently in use. In this article we suggest that instead of allowing computational predictions to drive investigation, it would be desirable, when possible, to systematically evaluate microRNA targets using inducible, stable, ectopic expression. The advantage of stable lines ectopically expressing microRNA(s) is that they allow an analysis of changes to both the proteome and the transcriptome. This would allow verification of targets, improve the design of prediction algorithms and greatly increase our understanding of the outcome of microRNA/mRNA interaction.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3092674PMC
http://dx.doi.org/10.4161/epi.6.3.14635DOI Listing
March 2011

Candidate dietary phytochemicals modulate expression of phase II enzymes GSTP1 and NQO1 in human lung cells.

J Nutr 2010 Aug 16;140(8):1404-10. Epub 2010 Jun 16.

Division of Pulmonary Medicine, Albert Einstein College of Medicine, Bronx, NY, USA.

Many phytochemicals possess cancer-preventive properties, some putatively through phase II metabolism-mediated mutagen/oxidant quenching. We applied human lung cells in vitro to investigate the effects of several candidate phytopreventive agents, including green tea extracts (GTE), broccoli sprout extracts (BSE), epigallocatechin gallate (EGCG), sulforaphane (SFN), phenethyl isothiocyanate (PEITC), and benzyl isothiocyanate (BITC), on inducing phase II enzymes glutathione S-transferase P1 (GSTP1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) at mRNA and protein levels. Primary normal human bronchial epithelial cells (NHBE), immortalized human bronchial epithelial cells (HBEC), and lung adenocarcinoma cells (A549) were exposed to diet-achievable levels of GTE and BSE (0.5, 1.0, 2.0 mg/L), or individual index components EGCG, SFN, PEITC, BITC (0.5, 1.0, 2.0 micromol/L) for 24 h, 48 h, and 6 d, respectively. mRNA assays employed RNA-specific quantitative RT-PCR and protein assays employed Western blotting. We found that in NHBE cells, while GSTP1 mRNA levels were slightly but significantly increased after exposure to GTE or BSE, NQO1 mRNA increased to 2- to 4-fold that of control when exposed to GTE, BSE, or SFN. Effects on NQO1 mRNA expression in HBEC cells were similar. NQO1 protein expression increased up to 11.8-fold in SFN-treated NHBE cells. Both GSTP1 and NQO1 protein expression in A549 cells were constitutively high but not induced under any condition. Our results suggest that NQO1 is more responsive to the studied chemopreventive agents than GSTP1 in human lung cells and there is discordance between single agent and complex mixture effects. We conclude that modulation of lung cell phase II metabolism by chemopreventive agents requires cell- and agent-specific discovery and testing.
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http://dx.doi.org/10.3945/jn.110.121905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2903300PMC
August 2010

Identification of carcinogen DNA adducts in human saliva by linear quadrupole ion trap/multistage tandem mass spectrometry.

Chem Res Toxicol 2010 Jul;23(7):1234-44

Division of Environmental Health Sciences, Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA.

DNA adducts of carcinogens derived from tobacco smoke and cooked meat were identified by liquid chromatography-electrospray ionization/multistage tandem mass spectrometry (LC-ESI/MS/MS(n)) in saliva samples from 37 human volunteers on unrestricted diets. The N-(deoxyguanosin-8-yl) (dG-C8) adducts of the heterocyclic aromatic amines 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-9H-pyrido[2,3-b]indole (AalphaC), 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), and the aromatic amine, 4-aminobiphenyl (4-ABP), were characterized and quantified by LC-ESI/MS/MS(n), employing consecutive reaction monitoring at the MS(3) scan stage mode with a linear quadrupole ion trap (LIT) mass spectrometer (MS). DNA adducts of PhIP were found most frequently: dG-C8-PhIP was detected in saliva samples from 13 of 29 ever-smokers and in saliva samples from 2 of 8 never-smokers. dG-C8-AalphaC and dG-C8-MeIQx were identified solely in saliva samples of three current smokers, and dG-C8-4-ABP was detected in saliva from two current smokers. The levels of these different adducts ranged from 1 to 9 adducts per 10(8) DNA bases. These findings demonstrate that PhIP is a significant DNA-damaging agent in humans. Saliva appears to be a promising biological fluid in which to assay DNA adducts of tobacco and dietary carcinogens by selective LIT MS techniques.
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http://dx.doi.org/10.1021/tx100098fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2916027PMC
July 2010

Smoking-Related Gene Expression in Laser Capture-Microdissected Human Lung.

Clin Cancer Res 2009 Dec;15(24):7562-7570

Authors' Affiliations: Division of Pulmonary Medicine, Department of Medicine, Department of Epidemiology and Population Health, and Department of Genetics, Albert Einstein College of Medicine, Bronx, New York; and Laboratory of Human Toxicology and Molecular Epidemiology, Wadsworth Center, New York State Department of Health, Albany, New York.

PURPOSE: Interindividual differences in quantitative expression could underlie a propensity for lung cancer. To determine precise individual gene expression signatures on a lung compartment-specific basis, we investigated the expression of carcinogen metabolism genes encoding cytochromes P450 (CYP) 1B1, 2A13, GSTP1, and a tumor suppressor gene p16 in laser capture-microdissected samples of human alveolar compartment (AC) and bronchial epithelial compartment (BEC) lung tissue from 62 smokers and nonsmokers. EXPERIMENTAL DESIGN: Tobacco exposure was determined by plasma nicotine, cotinine, and smoking history. Precise mRNA expression was determined using our RNA-specific qRT-PCR strategy, and correlated with detailed demographic and clinical characteristics. RESULTS: Several correlations of mRNA expression included (a) CYP1B1 in AC (positively with plasma nicotine level, P = 0.008; plasma cotinine level, P = 0.001), (b) GSTP1 in AC (positively with plasma cotinine level, P = 0.003), and (c) GSTP1 in BEC (negatively with smoke dose, P = 0.043; occupational risk, P = 0.019). CYP2A13 was rarely expressed in AC and not expressed in BEC. p16 expression was not correlated with any measured factor. For each gene, subjects showed expression that was individually concordant between these compartments. No clear association of mRNA expression with lung cancer risk was observed in this pilot analysis. CONCLUSIONS: The association between lung mRNA expression and tobacco exposure implies that gene-tobacco interaction is a measurable quantitative trait, albeit with wide interindividual variation. Gene expression tends to be concordant for alveolar and bronchial compartments for these genes in an individual, controlling for proximate tobacco exposure. (Clin Cancer Res 2009;15(24):7562-70).
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http://dx.doi.org/10.1158/1078-0432.CCR-09-1694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4238890PMC
December 2009

Gene promoter methylation assayed in exhaled breath, with differences in smokers and lung cancer patients.

Respir Res 2009 Sep 25;10:86. Epub 2009 Sep 25.

Wadsworth Center, Human Toxicology & Molecular Epidemiology, Albany, NY, USA.

Background: There is a need for new, noninvasive risk assessment tools for use in lung cancer population screening and prevention programs.

Methods: To investigate the technical feasibility of determining DNA methylation in exhaled breath condensate, we applied our previously-developed method for tag-adapted bisulfite genomic DNA sequencing (tBGS) for mapping of DNA methylation, and adapted it to exhaled breath condensate (EBC) from lung cancer cases and non-cancer controls. Promoter methylation patterns were analyzed in DAPK, RASSF1A and PAX5beta promoters in EBC samples from 54 individuals, comprised of 37 controls [current- (n = 19), former- (n = 10), and never-smokers (n = 8)] and 17 lung cancer cases [current- (n = 5), former- (n = 11), and never-smokers (n = 1)].

Results: We found: (1) Wide inter-individual variability in methylation density and spatial distribution for DAPK, PAX5beta and RASSF1A. (2) Methylation patterns from paired exhaled breath condensate and mouth rinse specimens were completely divergent. (3) For smoking status, the methylation density of RASSF1A was statistically different (p = 0.0285); pair-wise comparisons showed that the former smokers had higher methylation density versus never smokers and current smokers (p = 0.019 and p = 0.031). For DAPK and PAX5beta, there was no such significant smoking-related difference. Underlying lung disease did not impact on methylation density for this geneset. (4) In case-control comparisons, CpG at -63 of DAPK promoter and +52 of PAX5beta promoter were significantly associated with lung cancer status (p = 0.0042 and 0.0093, respectively). After adjusting for multiple testing, both loci were of borderline significance (p(adj) = 0.054 and 0.031). (5) The DAPK gene had a regional methylation pattern with two blocks (1) approximately -215--113 and (2) -84-+26; while similar in block 1, there was a significant case-control difference in methylation density in block 2 (p = 0.045); (6)Tumor stage and histology did not impact on the methylation density among the cases. (7) The results of qMSP applied to EBC correlated with the corresponding tBGS sequencing map loci.

Conclusion: Our results show that DNA methylation in exhaled breath condensate is detectable and is likely of lung origin. Suggestive correlations with smoking and lung cancer case-control status depend on individual gene and CpG site examined.
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http://dx.doi.org/10.1186/1465-9921-10-86DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2759916PMC
September 2009

Exhaled breath condensate appears to be an unsuitable specimen type for the detection of influenza viruses with nucleic acid-based methods.

J Virol Methods 2010 Jan 3;163(1):144-6. Epub 2009 Sep 3.

Laboratory of Viral Diseases, Wadsworth Center, New York State Department of Health, Albany, NY 12201-0509, USA.

Exhaled breath condensate is an airway-derived specimen type that has shown significant promise in the diagnosis of asthma, cancer, and other disorders. The presence of human genomic DNA in this sample type has been proven, but there have been no reports on its utility for the detection of respiratory pathogens. The suitability of exhaled breath condensate for the detection of influenza virus was investigated, as an indication of its potential as a specimen type for respiratory pathogen discovery work. Matched exhaled condensates and nasopharyngeal swabs were collected from 18 adult volunteers. Eleven cases were positive for influenza A virus, and one was positive for influenza B virus. All swab samples tested positive in real-time amplification assays, but only one exhaled condensate, an influenza A positive sample with a very high viral load, tested positive in the real-time RT-PCR assay. Most of the positive nasopharyngeal swab samples inoculated for virus culture also tested positive, whereas influenza virus was not grown from any of the exhaled condensate specimens. It was concluded that influenza viruses are not readily detectable with culture or nucleic acid-based techniques in this sample type, and that exhaled breath condensate may not be suitable for respiratory pathogen investigations with molecular methods.
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http://dx.doi.org/10.1016/j.jviromet.2009.08.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3730442PMC
January 2010

Haplotype-tagging single nucleotide polymorphisms in the GSTP1 gene promoter and susceptibility to lung cancer.

Cancer Detect Prev 2009 17;32(5-6):403-15. Epub 2009 Mar 17.

Laboratory of Human Toxicology and Molecular Epidemiology, Wadsworth Center, New York State Department of Health, Albany, NY, USA.

Background: Glutathione S-transferase (GST) P1 is a major phase II xenobiotic-metabolizing enzyme in the human lung. Our laboratory had previously identified nine single nucleotide polymorphisms (SNPs) in the GSTP1 gene promoter, which were then grouped into three main haplotypes (Hap1, Hap2, and Hap3) based on statistical inference. Hap3 was found to display a high expression phenotype. The main objective of the current study was to test the association between GSTP1 promoter haplotypes with the risk of lung cancer after determining the promoter haplotypes experimentally through cloning and sequencing.

Methods: We conducted a case-control analysis of 150 subjects with lung cancer and 329 controls with no personal history of the disease. The three statistically inferred GSTP1 promoter haplotypes were confirmed experimentally through cloning and sequencing. Haplotype-tagging SNPs were selected and GSTP1 haplotypes were tested for genetic association to lung cancer using unconditional logistic regression after adjusting for confounders. Statistical interaction between GSTP1 promoter haplotypes with either cigarette smoking or dietary fruit and vegetable intake were tested using the likelihood ratio test.

Results: We did not find protective effects of Hap3 against lung cancer, despite an adequately powered design for this main effect. Homozygous variants of tagSNPs -1738 T>A and -354 G>T, which tag Hap2, showed an increased (but statistically non-significant) risk of lung cancer among all subjects as well as among individuals with low fruit and vegetable intake, compared to homozygous wildtypes for these SNPs. We did not find significant interactions between Hap2 and dietary intake of fruits and vegetables.

Conclusions: Our results do not support significant main and modifying effects for GSTP1 promoter haplotypes on susceptibility to lung cancer in this population, but reinforce the protective effects of dietary intake of fruits and vegetables.
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http://dx.doi.org/10.1016/j.cdp.2009.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3730463PMC
August 2009

Dietary chemoprevention strategies for induction of phase II xenobiotic-metabolizing enzymes in lung carcinogenesis: A review.

Lung Cancer 2009 Aug 31;65(2):129-37. Epub 2009 Jan 31.

Division of Pulmonary Medicine, Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, United States.

Lung cancer is the leading cause of cancer mortality for men and women in the United States and is a growing worldwide problem. Protection against lung cancer is associated with higher dietary intake of fruits and vegetables, according to recent large epidemiologic studies. One strategy for lung cancer chemoprevention focuses on the use of agents to modulate the metabolism and disposition of tobacco, environmental and endogenous carcinogens through upregulation of detoxifying phase II enzymes. We summarize the substantial evidence that suggests that induction of phase II enzymes, particularly the glutathione S-transferases, plays a direct role in chemoprotection against lung carcinogenesis. The engagement of the Keap1-Nrf2 complex regulating the antioxidant response element (ARE) signaling pathway has been identified as a key molecular target of chemopreventive phase II inducers in several systems. Monitoring of phase II enzyme induction has led to identification of novel chemopreventive agents such as the isothiocyanate sulforaphane, and the 1,2-dithiole-3-thiones. However, no agents have yet demonstrated clear benefit in human cell systems, or in clinical trials. Alternative strategies include: (a) using intermediate cancer biomarkers for the endpoint in human trials; (b) high-throughput small molecule discovery approaches for induced expression of human phase II genes; and (c) integrative approaches that consider pharmacogenetics, along with pharmacokinetics and pharmacodynamics in target lung tissue. These approaches may lead to a more effective strategy of tailored chemoprevention efforts using compounds with proven human activity.
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http://dx.doi.org/10.1016/j.lungcan.2009.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3730487PMC
August 2009

Screening for DNA adducts by data-dependent constant neutral loss-triple stage mass spectrometry with a linear quadrupole ion trap mass spectrometer.

Anal Chem 2009 Jan;81(2):809-19

Division of Environmental Health Sciences, Wadsworth Center, New York State Department of Health, Albany, New York 12201, USA.

A two-dimensional linear quadrupole ion trap mass spectrometer (LIT/MS) was employed to simultaneously screen for DNA adducts of environmental, dietary, and endogenous genotoxicants, by data-dependent constant neutral loss scanning followed by triple-stage mass spectrometry (CNL-MS3). The loss of the deoxyribose (dR) from the protonated DNA adducts ([M + H - 116]+) in the MS/MS scan mode triggered the acquisition of MS3 product ion spectra of the aglycone adducts [BH2]+. Five DNA adducts of the tobacco carcinogen 4-aminobiphenyl (4-ABP) were detected in human hepatocytes treated with 4-ABP, and three DNA adducts of the cooked-meat carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) were identified in the livers of rats exposed to MeIQx, by the CNL-MS3 scan mode. Buccal cell DNA from tobacco smokers was screened for DNA adducts of various classes of carcinogens in tobacco smoke including 4-ABP, 2-amino-9H-pyrido[2,3-b]indole (AalphaC), and benzo[a]pyrene (BaP); the cooked-meat carcinogens MeIQx, AalphaC, and 2-amino-1-methyl-6-phenylmidazo[4,5-b]pyridine (PhIP); and the lipid peroxidation products acrolein (AC) and trans-4-hydroxynonenal (HNE). The CNL-MS3 scanning technique can be used to simultaneously screen for multiple DNA adducts derived from different classes of carcinogens, at levels of adduct modification approaching 1 adduct per 108 unmodified DNA bases, when 10 microg of DNA is employed for the assay.
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http://dx.doi.org/10.1021/ac802096pDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2646368PMC
January 2009

Overexpression of the microRNA hsa-miR-200c leads to reduced expression of transcription factor 8 and increased expression of E-cadherin.

Cancer Res 2007 Sep;67(17):7972-6

Ordway Research Institute, Albany, NY 12208, USA.

MicroRNAs are approximately 22-nucleotide sequences thought to interact with multiple mRNAs resulting in either translational repression or degradation. We previously reported that several microRNAs had variable expression in mammalian cell lines, and we examined one, miR-200c, in more detail. A combination of bioinformatics and quantitative reverse transcription-PCR was used to identify potential targets and revealed that the zinc finger transcription factor transcription factor 8 (TCF8; also termed ZEB1, deltaEF1, Nil-2-alpha) had inversely proportional expression levels to miR-200c. Knockout experiments using anti-microRNA oligonucleotides increased TCF8 levels but with nonspecific effects. Therefore, to investigate target predictions, we overexpressed miR-200c in select cells lines. Ordinarily, the expression level of miR-200c in non-small-cell lung cancer A549 cells is low in contrast to normal human bronchial epithelial cells. Stable overexpression of miR-200c in A549 cells results in a loss of TCF8, an increase in expression of its regulatory target, E-cadherin, and altered cell morphology. In MCF7 (estrogen receptor-positive breast cancer) cells, there is endogenous expression of miR-200c and E-cadherin but TCF8 is absent. Conversely, MDA-MB-231 (estrogen receptor-negative) cells lack detectable miR-200c and E-cadherin (the latter reportedly due to promoter region methylation) but express TCF8. The ectopic expression of miR-200c in this cell line also reduced levels of TCF8, restored E-cadherin expression, and altered cell morphology. Because the down-regulation of E-cadherin is a crucial event in epithelial-to-mesenchymal transition, loss of miR-200c expression could play a significant role in the initiation of an invasive phenotype, and, equally, miR-200c overexpression holds potential for its reversal.
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http://dx.doi.org/10.1158/0008-5472.CAN-07-1058DOI Listing
September 2007

Validity of messenger RNA expression analyses of human saliva.

Clin Cancer Res 2006 Sep;12(17):5033-9

Laboratory of Human Toxicology and Molecular Epidemiology, Wadsworth Center, New York State Department of Health, Albany, New York 12201-0509, USA.

Purpose: The origins of expression microarray and reverse transcription-PCR (RT-PCR) signals in human saliva were evaluated.

Experimental Design: The "RNA" extracts from human saliva samples were treated with vehicle, DNase, or RNase. Two-step amplification and hybridization to Affymetrix 133A cDNA microarrays were then done. Confirmatory RT-PCR experiments used conventionally designed PCR primer pairs for the reference housekeeper transcripts encoding 36B4, beta-actin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA sequences, which are known to be homologous to genomic DNA pseudogene sequences. Negative controls included the omission of reverse transcriptase ("no-RT") to detect any DNA-derived signal. Finally, an RNA-specific RT-PCR strategy eliminated confounding signals from contaminating genomic DNA.

Results: Microarray experiments revealed that untreated, DNase-treated, and RNase-treated "RNA" extracts from saliva all yielded negligible overall signals. Specific microarray signals for 36B4, beta-actin, and GAPDH were low, and were unaffected by RNase. Real-time quantitative RT-PCR reactions using conventional, non-RNA-specific primers on saliva samples yielded PCR products for 36B4, beta-actin, and GAPDH; DNase-treated saliva samples did not yield a PCR product, and the "no-RT" and "+RT" conditions yielded similar amounts of PCR product. The RNA-specific RT-PCR strategy, across all conditions, yielded no PCR product from saliva.

Conclusions: The combination of (a) a minimal microarray signal, which was unaffected by RNase treatment, (b) the presence of a conventional RT-PCR housekeeper product in both RNase-treated and no-RT saliva samples, (c) the absence of a conventional RT-PCR housekeeper product in DNase-treated conditions, and (d) the absence of a RNA-specific RT-PCR product shows that any microarray or RT-PCR signal in the saliva must arise from genomic DNA, not RNA. Thus, saliva extracts do not support mRNA expression studies.
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http://dx.doi.org/10.1158/1078-0432.CCR-06-0501DOI Listing
September 2006

Potential mRNA degradation targets of hsa-miR-200c, identified using informatics and qRT-PCR.

Cell Cycle 2006 Sep 1;5(17):1951-6. Epub 2006 Sep 1.

Ordway Research Institute, Albany, New York 12208, USA.

Using an anchored oligo(dT) based RT-PCR approach we quantified endogenous expression of ten microRNAs in six cell lines. This identified a miRNA, miR-200c, with variable expression, ranging from undetectable in MDA-MB-231 and HT1080 to highly expressed in MCF7. The variable expression provided a model system to investigate endogenous interactions between miRNAs and their computationally predicted targets. As the expression level of the predicted mRNA targets and miR-200c in these lines should have an inverse relationship if cleavage or degradation results from the interaction. To select targets for analysis we used Affymetrix expression data and computational prediction programs. Affymetrix data indicated approximately 3500 candidate mRNAs, absent in MCF7 and present in MDA-MB-231 or HT1080. These targets were cross-referenced against approximately 600 computationally predicted miR-200c targets, identifying twenty potential mRNAs. Expression analysis by qRT-PCR of these targets and an additional ten mRNAs (selected using the prediction program ranking alone) revealed four mRNAs, BIN1, TCF8, RND3 and LHFP with an inverse relationship to miR-200c. Of the remainder, the majority did not appear to be degraded (and may be translational targets) or were undetectable in the cell lines examined. Finally, inhibition of miR-200c using an anti-miRNA 2'-0-Methyl oligonucleotide (AMO) resulted in an increase in expression of one of the targets, the transcription factor TCF8. These results indicate that a single miRNA could directly affect the mRNA levels of an important transcription factor, albeit in a manner specific to cell lines. Further investigation is required to confirm this in vivo and determine any translational effects.
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http://dx.doi.org/10.4161/cc.5.17.3133DOI Listing
September 2006

DNA methylation mapping by tag-modified bisulfite genomic sequencing.

Anal Biochem 2006 Aug 2;355(1):50-61. Epub 2006 Jun 2.

Laboratory of Human Toxicology and Molecular Epidemiology, Wadsworth Center, NYS Department of Health, Albany, NY 12201, USA.

A tag-modified bisulfite genomic sequencing (tBGS) method employing direct cycle sequencing of polymerase chain reaction (PCR) products at kilobase scale, without conventional DNA fragment cloning, was developed for simplified evaluation of DNA methylation sites. The method entails subjecting bisulfite-modified genomic DNA to a second-round PCR amplification employing GC-tagged primers. Qualitative results from tBGS closely correlated with those from conventional BGS (R=0.935, p=0.002). In application, the intertissue and interindividual CpG methylation differences in promoter sequence for two genes, CYP1B1 and GSTP1, were then explored across four human tissue types (peripheral blood cells, exfoliated buccal cells, paired nontumor-tumor lung tissues), and two lung cell types in culture (normal NHBE and malignant A549). Predominantly conserved methylation maps for the two gene promoters were apparent across donors and tissues. At any given CpG site, variation in the degree of methylation could be determined by the relative height of C and T peaks in the sequencing trace. Methylation maps for the GSTP1 promoter diverged between NHBE (unmethylated) and A549 (completely methylated) cells in a previously unexplored upstream region, correlating with a 2.7-fold difference in GSTP1 mRNA expression (p<0.01). The tBGS method simplifies detailed methylation scanning of kilobase-scale genomic DNA, facilitating more ambitious genomic methylation mapping studies.
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http://dx.doi.org/10.1016/j.ab.2006.05.010DOI Listing
August 2006

Haplotype-environment interactions that regulate the human glutathione S-transferase P1 promoter.

Cancer Res 2006 Jun;66(12):6439-48

Laboratory of Human Toxicology and Molecular Epidemiology, Wadsworth Center, New York State Department of Health, NY, USA.

Phase II detoxification of carcinogens is reported to mediate some of the anticarcinogenesis effects of candidate chemopreventive agents. We explored the interaction between sequence variation in the GSTP1 gene promoter and candidate chemopreventive exposure in regulating human GSTP1 expression. Polymorphisms along 1.8 kb of the GSTP1 promoter were identified in leukocytes [peripheral blood mononuclear cells (PBMC)] from 40 Caucasian subjects. Ten promoter polymorphisms (9 previously unreported) displayed strong linkage disequilibrium, yielding identification of three frequently observed haplotypes [HAP1 (43%), HAP2 (36%), and HAP3 (8%)]. Each haplotype was cloned into luciferase reporter constructs and transfected into normal human bronchial epithelial cells. Basal HAP3 reporter activity was significantly elevated (1.8-fold) but decreased to the same levels as HAP2 and HAP1 with increasing concentrations of sulforaphane, benzyl isothiocyanate (BITC), and epigallocatechin gallate (EGCG). To confirm native HAP3 functionality, we quantitated mRNA expression in uncultured PBMCs and in laser microdissected normal lung epithelial cells (MNLEC) from the same patients. Basal mRNA expression was higher in HAP3 individuals [1.8-fold (PBMC) and 4-fold (MNLEC) for HAP3 heterozygotes and 2.3-fold (PBMC), and 15-fold (MNLEC) for the HAP3 homozygote] than in the other genotypes. PBMC GSTP1 mRNA expression correlated to MNLEC expression (R2 = 0.77). After culture and in vitro exposure to sulforaphane, BITC, or EGCG, the elevated GSTP1 mRNA expression of PBMCs from HAP3 individuals decreased to common expression levels. Elevated HAP3 function was confirmed at the protein level in PBMCs (5-fold higher for HAP3 heterozygotes and 7.6-fold for the HAP3 homozygote). These data suggest a potentially protective GSTP1 promoter haplotype and unpredicted inhibitory chemopreventive agent-haplotype interactions.
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http://dx.doi.org/10.1158/0008-5472.CAN-05-4457DOI Listing
June 2006

Exfoliated buccal and microdissected lung cell expression of antioxidant enzymes.

Cancer Detect Prev 2005 10;29(6):552-61. Epub 2005 Nov 10.

Laboratory of Human Toxicology and Molecular Epidemiology, Wadsworth Center, New York State Department of Health, P.O. Box 509, Albany, NY 12201-0509, USA.

Introduction: An exfoliated buccal cell biomarker assay for antioxidant gene transcript levels was used to measure inter-tissue concordance with lung, and inter-subject variability in a lung cancer case-control study.

Methods: First, qualitative RNA-specific RT-PCR was used to compare expression in exfoliated buccal cells with that in laser microdissected lung tissue remote from the tumor from 14 individuals providing both specimens.

Results: There was complete [100% for quinone oxidoreductase 1 (NQO1), glutathione peroxidase (GPX), and superoxide dismutase 1 (SOD1)], or predominant [85.7% for catalase (CAT)] inter-tissue concordance for qualitative expression. Second, quantitative real-time RT-PCR for antioxidant enzyme transcript levels was performed in exfoliated buccal samples from these same 14 individuals, as well as 28 additional individuals providing buccal cells only, for a total of 42 buccal specimens (19 current smokers and 23 ex- or never-smokers), of whom 26 (61.39%) had a new diagnosis of lung cancer.

Discussion: Wide inter-individual expression differences for each gene transcript (>10(1)-10(4)-fold) were observed in the exfoliated buccal cells, unrelated to smoking and case-control status. In multivariate analyses, family history of tobacco-related malignancy correlated inversely with buccal NQO1 and CAT mRNA levels (p=0.003, p<0.001, respectively). This antioxidant expression trait may relate to family risk of cancer, but is notably unrelated to oxidant challenges inherent in cigarette smoke.
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http://dx.doi.org/10.1016/j.cdp.2005.09.003DOI Listing
March 2006

Gene-environment interaction signatures by quantitative mRNA profiling in exfoliated buccal mucosal cells.

Cancer Res 2004 Sep;64(18):6805-13

Laboratory of Human Toxicology and Molecular Epidemiology, New York State Department of Health, Albany, NY 12201-0509, USA.

Exfoliated cytologic specimens from mouth (buccal) epithelium may contain viable cells, permitting assay of gene expression for direct and noninvasive measurement of gene-environment interactions, such as for inhalation (e.g., tobacco smoke) exposures. We determined specific mRNA levels in exfoliated buccal cells collected by cytologic brush, using a recently developed RNA-specific real-time quantitative reverse transcription-PCR strategy. In a pilot study, metabolic activity of exfoliated buccal cells was verified by 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium assay in vitro. Transcriptional activity was observed, after timed in vivo exposure to mainstream tobacco smoke resulted in induction of CYP1B1 in serially collected buccal samples from the one subject examined. For a set of 11 subjects, mRNA expression of nine genes encoding carcinogen- and oxidant-metabolizing enzymes qualitatively detected in buccal cells was then shown to correlate with that in laser-microdissected lung from the same individuals (Chi2 = 52.91, P < 0.001). Finally, quantitative real-time reverse transcription-PCR assays for seven target gene (AhR, CYP1A1, CYP1B1, GSTM1, GSTM3, GSTP1, and GSTT1) and three reference gene [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin, and 36B4] transcripts were performed on buccal specimens from 42 subjects. In multivariate analyses, gender, tobacco smoke exposure, and other factors were associated with the level of expression of CYP1B1, GSTP1, and other transcripts on a gene-specific basis, but substantial interindividual variability in mRNA expression remained unexplained. Within the power limits of this pilot study, gene expression signature was not clearly predictive of lung cancer case or control status. This noninvasive and quantitative method may be incorporated into high-throughput human applications for probing gene-environment interactions associated with cancer.
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http://dx.doi.org/10.1158/0008-5472.CAN-04-1771DOI Listing
September 2004

Buccal-lung comparison of quantitative expression of carcinogen and oxidant metabolism genes in human subjects.

Chest 2004 May;125(5 Suppl):107S-8S

New York State Department of Health, State University of New York School of Public Health, and Albany Medical College, Pulmonary and Critical Care Medicine, Albany, NY 12201-0509, USA.

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http://dx.doi.org/10.1378/chest.125.5_suppl.107s-aDOI Listing
May 2004