Effect of titanium dioxide nanoparticles on histone modifications and histone modifying enzymes expression in human cell lines.

Titanium dioxide (TiO2) nanoparticles are widely manufactured, with a range of applications in consumer products. Significant toxicity of TiO2 nanoparticles has, however, been recognized, suggesting considerable risk to human health. To evaluate fully their toxicity, assessment of the epigenetic action of these nanoparticles is critical. However, only few studies are available examining the capability of nanoparticles to alter epigenetic integrity. In the present study, the effect of TiO2 nanoparticles exposure on histone modifications, a major epigenetic mechanism, was investigated in human colorectal (Caco-2) and lung (NL20) epithelial cell lines. Histone H3 and H4 modifications were assessed by array analysis using the EpiQuickTM Histone H3 or H4 Modification Multiplex Assay. Seventeen histone modifications were identified with altered levels after exposure to TiO2 nanoparticles. Changes in several selected histone modifications (Caco-2 cells: H3cit, H3K9me3, H3K27me3, H3K36me3, H3K9ac, and H4K8ac; NL20 cells: H3K4me3, H3K9me3, H3K27me3, H3K9ac, and H3K18ac) were verified by Western blot analysis. The results also revealed aberrant expression of histone modifying enzymes in TiO2 exposed cells. Expression levels were determined by array analysis using the Human Epigenetic Chromatin Modification Enzymes RT2 Profiler™ PCR Array, with 12 genes identified in both Caco-2 cells and NL20 cells. qRT-PCR analysis confirmed the array results for several selected histone modifying enzyme genes (ASH1L, CARM1, EHMT2, HAT1, HDAC9, KMT2E, NCOA1, SETDB2, and USP16). The findings from this study clearly demonstrate the impact of TiO2 nanoparticles exposure on histone modification in two human cell lines, supporting potential involvement of this epigenetic mechanism in the toxicity of TiO2 nanoparticles. Hence, for complete assessment of potential risk from nanoparticle exposure, epigenetic studies are critical.

Modulation of Estrogen α and Progesterone Receptors in Triple Negative Breast Cancer Cell Lines: The Effects of Vorinostat and Indole-3-Carbinol In Vitro.

BACKGROUND/AIM: Triple negative cancer (TNBC) is a subtype of breast cancer that is highly aggressive, with poor prognosis and responds differently to treatments. This study investigated the role of vorinostat and indole-3-carbinol (I3C) on regulating critical receptors that are not normally expressed in TNBC. MATERIALS AND METHODS: Using real-time PCR, immunostaining, and western blots, the re-expression of estrogen receptor α (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2) receptors was examined in four different TNBC cell types. RESULTS: ERα was re-expressed in three subtypes using vorinostat and I3C. Re-expression of the PR by vorinostat was also detected. Neither vorinostat nor I3C resulted in re-expression of the HER2 receptor. A significant decrease in growth and sensitivity to tamoxifen was also noted. CONCLUSION: The results of this study show that vorinostat and I3C modulate the re-expression of critical receptors in certain subtypes of TNBC through several pathways and these effects can be influenced by the molecular profiles of TNBCs.

Effect of titanium dioxide nanoparticles on DNA methylation in multiple human cell lines.

Nanoscale titanium dioxide (TiO2) is manufactured in wide scale, with a range of applications in consumer products. Significant toxicity of TiO2 nanoparticles has, however, been recognized, suggesting considerable risk to human health. To evaluate fully their toxicity, assessment of the epigenetic action of these nanoparticles is critical. However, only few studies are available examining capability of nanoparticles to alter epigenetic integrity. In the present study, the effect of TiO2 nanoparticles exposure on DNA methylation, a major epigenetic mechanism, was investigated in in vitro cellular model systems. A panel of cells relevant to portals of human exposure (Caco-2 (colorectal), HepG2 (liver), NL20 (lung), and A-431 (skin)) was exposed to TiO2 nanoparticles to assess effects on global methylation, gene-specific methylation, and expression levels of DNA methyltransferases, MBD2, and UHRF1. Global methylation was determined by enzyme-linked immunosorbent assay-based immunochemical analysis. Degree of promoter methylation across a defined panel of genes was evaluated using EpiTect Methyl II Signature PCR System Array technology. Expression of DNMT1, DNMT3a, DNMT3b, MBD2, and URHF1 was quantified by qRT-PCR. Decrease in global DNA methylation in cell lines Caco-2, HepG2, and A-431 exposed to TiO2 nanoparticles was shown. Across four cell lines, eight genes (CDKN1A, DNAJC15, GADD45A, GDF15, INSIG1, SCARA3, TP53, and BNIP3) were identified in which promotors were methylated after exposure. Altered expression of these genes is associated with disease etiology. The results also revealed aberrant expression of epigenetic regulatory genes involved in DNA methylation (DNMT1, DNMT3a, DNMT3b, MBD2, and UHRF1) in TiO2 exposed cells, which was cell type dependent. Findings from this study clearly demonstrate the impact of TiO2 nanoparticles exposure on DNA methylation in multiple cell types, supporting potential involvement of this epigenetic mechanism in the toxicity of TiO2 nanoparticles. Hence for complete assessment of potential risk from nanoparticle exposure, epigenetic studies are critical.

Epigenome-wide association study of peripheral blood mononuclear cells in systemic lupus erythematosus: Identifying DNA methylation signatures associated with interferon-related genes based on ethnicity and SLEDAI.

Systemic lupus erythematosus (SLE or lupus) is a heterogeneous autoimmune disease characterized by the involvement of multiple organs and the production of antinuclear antibodies. DNA methylation plays an important role in the pathogenesis of lupus. We have performed an epigenome-wide DNA methylation study in lupus and healthy control (non-lupus) subjects to identify epigenetic patterns in lupus characterized ethnicity and SLE disease activity index (SLEDAI). A total of fifty-seven lupus patients (39 African American (AA) and 18 European American (EA)) and 33 healthy controls (17 AA and 16 EA) were studied. Differential DNA methylation between lupus patients and controls was assessed for approximately 485,000 CpG sites across the genome. We identified 41 differentially methylated sites (associated with 30 genes) between lupus and control s subjects, 85% of which were hypomethylated. Significant hypomethylation of differentially methylated sites was associated with several interferon-related genes, including MX1, IFI44L, PARP9, DT3XL, IFIT1, IFI44, RSAD2, PLSCR1, and IRF7. Several of these associated genes were also hypomethylated in comparisons between AA lupus and AA non-lupus subjects and between lupus patients with SLEDAI>6 and non-lupus subjects. Our analysis of gene expression data through RT-PCR confirmed these findings. Thus, the results indicate epigenetics susceptibility in lupus, which may be associated with SLEDAI score and ethnicity. In addition, our findings support the importance of the Type 1 interferon pathway in lupus pathogenesis.

In vitro analysis of factors influencing CYP1A2 expression as potential determinants of interindividual variation.

Individual differences in drug metabolism contribute to interindividual variation that characterizes responses to drugs and risk in exposure to foreign chemicals. Large individual differences are found in expression levels of CYP1A2, a major drug-metabolizing enzyme. Underlying causes for this variation are not well understood. Several factors, including tobacco smoking, consumption of cruciferous vegetables, and sex, have been associated with modulating CYP1A2 expression. To understand factors regulating expression of CYP1A2 in establishing a causal relationship, this study examined effects of cigarette smoke condensate (CSC), indole-3-carbinol (I3C), and 17ß-estradiol (estradiol) on CYP1A2 expression in in vitro systems using human liver and lung cells. Treatment with CSC (2-25 µg/mL) significantly increased levels of CYP1A2 in six cell lines examined, in a concentration- and time-dependent manner. Fold changes in expression levels relative to controls varied among cell lines. CYP1A2 enzymatic activity also increased with CSC exposure. Treatment of H1299 and HepB3 cells with dietary agent I3C (50 and 100 µmol/L) increased CYP1A2 expression. In human cell lines H1299 and H1395, treatment with estradiol (10 and 100 nmol/L) significantly reduced expression of CYP1A2. Using ChIP assays, effects of CSC on histone modifications were analyzed. Increases in H3K4me3 and H4K16ac were observed at several segments in the CYP1A2 gene, whereas H3K27me3 decreased, following CSC treatment. These results suggest that CYP1A2 expression is affected epigenetically by CSC. Additional studies will be needed to further establish regulatory mechanisms underlying effects of various environmental, dietary, and endogenous factors on CYP1A2 expression in better predicting individual variation.

Evaluation of Batch Variations in Induced Pluripotent Stem Cell-Derived Human Cardiomyocytes from 2 Major Suppliers.

Drug-induced proarrhythmia is a major safety issue in drug development. Developing sensitive in vitro assays that can predict drug-induced cardiotoxicity in humans has been a challenge of toxicology research for decades. Recently, induced pluripotent stem cell-derived human cardiomyocytes (iPSC-hCMs) have become a promising model because they largely replicate the electrophysiological behavior of human ventricular cardiomyocytes. Patient-specific iPSC-hCMs have been proposed for personalized cardiac drug selection and adverse drug response prediction; however, many procedures are involved in cardiomyocytes differentiation and purification process, which may result in large line-to-line and batch-to-batch variations. Here, we examined the purity, cardiac ion channel gene expression profile, and electrophysiological response of 3 batches of iPSC-hCMs from each of 2 major cell suppliers. We found that iPSC-hCMs from both vendors had similar purities. Most of the cardiac ion channel genes were expressed uniformly among different batches of iCells, while larger variations were found in Cor.4U cells, particularly in the expression of CACNA1C, KCND2, and KCNA5 genes, which could underlie the differences in baseline beating rate (BR) and field potential duration (FPD) measurements. Although, in general, the electrophysiological responses of different batches of cells to Na+, Ca2+, Ikr, and Iks channel blockers were similar, with Ikr blocker-induced proarrhythmia, the sensitivities were depended on baseline BR and FPD values: cells that beat slower had longer FPD and greater sensitivity to drug-induced proarrhythmia. Careful evaluation of the performance of iPSC-hCMs and methods of data analysis is warranted for shaping regulatory standards in qualifying iPSC-hCMs for drug safety testing.

Prolactin and Dehydroepiandrosterone Levels in Women with Systemic Lupus Erythematosus: The Role of the Extrapituitary Prolactin Promoter Polymorphism at -1149G/T.

Systemic lupus erythematosus (SLE) has shown an association with high levels of prolactin, low levels of dehydroepiandrosterone (DHEA), and induction of inflammatory cytokines in the serum of patients with the disease. This preliminary study examined the relevance of a -1149G/T functional single-nucleotide polymorphism (SNP) (rs1341239) in the promoter of the extrapituitary prolactin gene in a cohort of African American and European American women with lupus. Examination of this SNP revealed that the -1149TT genotype was correlated with higher levels of prolactin in serum and prolactin gene expression (p = 0.0001) in peripheral blood mononuclear cells (PBMCs). Lower levels of DHEA in serum were demonstrated in lupus patients (p = 0.001); those with the -1149TT genotype had the lowest levels of DHEA. Furthermore, a small subset of women who were on DHEA therapy and had a TT genotype showed a significant decrease in prolactin gene expression and lower disease activity scores (SLEDAI). Lupus patients, particularly African Americans, had significantly higher levels of IL-6 (p = 0.0001) and TNF-α (p = 0.042). This study suggests that the -1149TT genotype may be a risk factor for lupus and may predict who could possibly benefit from DHEA therapy; therefore, these results should be validated in a larger cohort with all ethnic groups.

Cytotoxicity of chronic exposure to 4 cigarette smoke condensates in 2 cell lines.

Tobacco use is the leading preventable cause of death. The cytotoxicity of cigarette smoke condensate (CSC), the particulate fraction of cigarette smoke without the vapor phase, has mostly been tested in short-term in vitro studies lasting from a few hours to a few days. Here, we assessed the toxicity of CSCs from 2 reference cigarettes, 3R4F and CM6, using a primary human small airway epithelial (PSAE) cell line by quantifying adenosine 5'-triphosphate (ATP), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), total glutathione (reduced glutathione [GSH] + oxidized glutathione [GSSG]), and lactate dehydrogenase (LDH) release over the course of 28 days. The CSCs, 0.3 to 10 µg/mL, promoted cell proliferation at 120 hours of exposure, but demonstrated cytotoxicity at days 14 and 28. Interestingly, CSCs, 0.3 to 3 µg/mL, showed a cell death effect at day 14 but induced cell proliferation at day 28. Consistently, transformation associated with morphological changes began by day 14 and the transformed cells grew dramatically at day 28. The LDH assay appeared to be sensitive for assessing early cell damage, whereas the ATP, MTS, and GSH assays were more suitable for determining later stage CSCs-induced cytotoxicity. The ATP assay showed greater sensitivity than the MTS and GSH assays. We also assessed the toxicity of CSCs in an human Telomerase Reverse Transcriptase (hTERT)-immortalized Barrett esophagus cell line (CP-C). The CP-C cells demonstrated dose- and time-dependent cytotoxicity over the course of 28 days but displayed higher resistance to CSCs than PSAE cells. This study demonstrates that CSCs cause cytotoxicity and induce transformation related to cell resistance and cell invasion properties.

Expression of drug transporters in human kidney: impact of sex, age, and ethnicity.

BACKGROUND: Differences in expression of drug transporters in human kidney contribute to changes in pharmacokinetics and toxicokinetics of a variety of drug compounds. The basal expression levels of genes involved in drug transport processes in the kidney introduces differences in bioavailability, distribution, and clearance of drugs, possibly influencing drug efficacy and adverse reactions. Sex differences in gene expression of transporters are a key cause of differences in sex-dependent pharmacokinetics, which may characterize many drugs and contribute to individual differences in drug efficacy and toxicity. Therefore, evaluating the expression of drug transporters in normal human kidneys is important to better understand differences in drug bioavailability, distribution, and clearance of drugs in humans. Other factors such as age and ethnicity may also contribute to individual differences in gene expression of drug transporters in the human kidney. METHODS: Quantitative real-time PCR (QRT-PCR) was performed to determine the gene expression of 30 drug transporters in 95 age-matched normal human kidney tissues. Multiple Student's t-tests (Sidak-Bonferroni correction) and two-way ANOVA (Bonferroni correction) analyses were used to determine statistically significant differences. RESULTS: In the 30 transporter genes examined, sex, ethnicity, and age differences in gene expression were exhibited in normal human kidney tissue. These changes in expression were not found to be differentially significant. However, sex-age and sex-ethnicity interactions were found to be statistically significant. For sex-age interactions, SCL22A12 was found to be significantly higher expressed in females <50 years compared to males <50 years. Expression levels of SLC22A2, SLC22A12, SLC6A16, and ABCB6 were significantly higher in females <50 years compared to females ≥50 years. In sex-ethnicity interactions, expression levels of ATP7B and KCNJ8 were found to be significantly higher in African American females compared to European American females. Also, the expression of SLC31A2 was significantly higher in European American males compared to European American females. CONCLUSIONS: Sex, age, and ethnic differences impacted the expression of drug transporters in normal human kidneys, which suggests that the analysis of gene expression of drug transporters will aid in improving the usage/dosage of drug therapies influencing personalized medicine and susceptibility to adverse drug reactions.

Cigarette smoke condensate and individual constituents modulate DNA methyltransferase expression in human liver cells.

OBJECTIVES: Previous studies found higher expression levels of DNA methyltransferase 1 in liver samples from smokers compared to those from non-smokers. In contrast, expression levels of DNA methyltransferase 3a and DNA methyltransferase 3b were similar in smokers and non-smokers. This study extends these studies to establish a causal linkage to cigarette smoke exposure by examining whether DNA methyltransferase expression is modulated by cigarette smoke condensate. METHODS: These experiments were conducted in an in vitro system using HepG2 human liver cells. The dose range of cigarette smoke condensate was 0.1-120 µg/mL. The duration of exposure was up to 72 h. RESULTS: In a 24-h exposure, DNA methyltransferase 1 expression was found to increase significantly in a dose-dependent manner (greater than threefold at 100 µg/mL cigarette smoke condensate). Expression levels of DNA methyltransferase 3a and DNA methyltransferase 3b were, however, not affected under these conditions. The effect of two cigarette constituents, nicotine and cotinine, on DNA methyltransferase 1 expression was also examined. Nicotine exposure significantly increased DNA methyltransferase 1 expression in a dose-dependent manner (greater than twofold at 50 µM). However, under these conditions, cotinine did not increase DNA methyltransferase 1 expression. CONCLUSION: These results clearly provide additional support of the modulating effect of cigarette smoke on DNA methyltransferase 1 expression. Given the potential of alterations in DNA methyltransferase expression to affect cellular function, this pathway may play a critical role in cigarette smoke-induced toxicity.

Effect of cigarette smoke condensate on gene promoter methylation in human lung cells.

BACKGROUND: In lung cancer, an association between tobacco smoking and promoter DNA hypermethylation has been demonstrated for several genes. However, underlying mechanisms for promoter hypermethylation in tobacco-induced cancer are yet to be fully established. METHODS: Promoter methylation was evaluated in control and cigarette smoke condensate (CSC) exposed human lung cells using the Methyl-Profiler DNA Methylation PCR System. PSAE cells were exposed to 0.3 or 1.0 µg/ml CSC for 72 hours and longer term for 14 and 30 days. NL-20 cells were exposed for 30 days to 10 or 100 µg/ml CSC. RESULTS: Promoters of several genes, including hsa-let-7a-3, CHD1, CXCL12, PAX5, RASSF2, and TCF21, were highly methylated (>90%); hsa-let-7a-3 was affected in both cell lines and under all exposure conditions. Level of methylation tended to increase with CSC concentration and exposure duration (statistical differences were not determined). Percentage methylation of TCF21, which was >98% at exposures of 10 or 100 µg/ml CSC, was found to be reduced to 28% and 42%, respectively, in the presence of the dietary agent genistein. CONCLUSIONS: Using array techniques, several tumor suppressor genes in human lung cells were identified that undergo promoter hypermethylation, providing further evidence of their potential involvement in tobacco smoke-induced lung carcinogenesis and their use as potential biomarkers of harm in tobacco smoke exposure. Results from the study also demonstrated the potential of a dietary agent to exert chemopreventive activity in human tissue against tobacco smoke related diseases through modulation of DNA methylation. Additional studies are needed to confirm these findings.

ATP-Binding Cassette Genes Genotype and Expression: A Potential Association with Pancreatic Cancer Development and Chemoresistance?

Genetic polymorphisms in ABC (ATP-binding cassette) transporter genes are associated with differential responses to chemotherapy in various cancers including pancreatic cancer. In this study, four SNPs in the ABCB1, ABCC1, and ABCG2 genes were investigated in normal and pancreatic cancerous specimens. The expression of the three transporters was also analyzed. The TT genotypes of G2677T and C3435T in ABCB1 gene were associated with lower risk of developing pancreatic cancer (P = 0.013, OR = 0.35 and P = 0.015, OR = 0.29, resp.). To our knowledge, this is the first report of the common polymorphisms in the ABCB1 gene affecting the genetic risk of developing pancreatic cancer. Moreover, the expression of ABCB1 in 2677TT and 3435TT carriers was lower compared to the wild-type homozygotes and heterozygotes. A cell viability assay, using standard pancreatic cancer cell lines, revealed that the ABCB1 2677TT-3455TT haplotype was more sensitive than the other haplotypes to gemcitabine. Conclusion. Polymorphisms in ABCB1 G2677T and G3435T were associated with differential susceptibility to pancreatic cancer and may predict responses to chemotherapy.

Increased expression of Toll-like receptors (TLRs) 7 and 9 and other cytokines in systemic lupus erythematosus (SLE) patients: ethnic differences and potential new targets for therapeutic drugs.

Increased expression of pro-inflammatory cytokines such as interferon, tumor necrosis factors (TNFs) and specific interleukins (ILs) has been found in a number of autoimmune diseases, including systemic lupus erythematous (SLE). These cytokines are induced by toll-like receptors (TLRs). Toll-like receptors are activated in response to accumulation of apoptotic bodies. These receptors play critical roles in innate immune systems. Increased levels of interferon-alpha (INF-α) have also been found in many SLE patients and often correlate with disease severity. The objectives of this study were to examine the expression of selected TLRs and cytokines that have been identified in animal models and some limited human studies in a group of African Americans (AA) and European Americans (EA) women with lupus in comparison to age-matched non-lupus women. Blood samples were consecutively obtained by informed consent from 286 patients, 153 lupus and 136 non-lupus, seen in the rheumatology clinics at East Carolina University. Cytokines were analyzed from blood serum using enzyme linked immunoassay (ELISA) for IL-6 and INF-α. Total RNA was isolated, using a Paxgene kit, from peripheral blood mononuclear cells of African American and European American women blood samples. Quantitative real-time PCR using the CFX real-time system was conducted on all samples to determine TLRs 7 and 9, as well as INF-α expression. Toll-like receptor 7 (p<0.01) and 9 (p=0.001) expression levels were significantly increased in lupus patients compared to age-matched controls. African American women with lupus had a 2-fold increase in TLR-9 expression level when compared to their healthy controls or European American lupus patients. However, there was no ethnic difference in expression of TLR-7 in lupus patients. INF-α expression was significantly higher in lupus patients (p<0.0001) and also showed ethnic difference in expression. Serum levels revealed significant increases in expression of IL-6, IFN-γ and TNF-α in lupus patients compared to non-lupus patients. African American women with lupus had significantly higher serum levels of IL-6 and TNF-α. African American women with lupus demonstrated increased levels of specific pro-inflammatory cytokines and Toll-like receptors when compared to EA women. Increased expression in these lupus patients provides an opportunity for targeting with antagonist as a new therapy for systemic lupus erythematous.

Restoration of the methylation status of hypermethylated gene promoters by microRNA-29b in human breast cancer: A novel epigenetic therapeutic approach.

It is well established that transcriptional silencing of critical tumor-suppressor genes by DNA methylation is a fundamental component in the initiation of breast cancer. However, the involvement of microRNAs (miRNAs) in restoring abnormal DNA methylation patterns in breast cancer is not well understood. Therefore, we investigated whether miRNA-29b, due to its complimentarity to the 3'- untranslated region of DNA methyltransferase 3A (DNMT3A) and DNMT3B, could restore normal DNA methylation patterns in human breast cancers and breast cancer cell lines. We demonstrated that transfection of pre-miRNA-29b into less aggressive MCF-7 cells, but not MDA-MB-231 mesenchymal cells, inhibited cell proliferation, decreased DNMT3A and DNMT3B messenger RNA (mRNA), and decreased promoter methylation status of ADAM23 , CCNA1, CCND2, CDH1, CDKN1C, CDKN2A, HIC1, RASSF1, SLIT2, TNFRSF10D, and TP73 tumor-suppressor genes. Using methylation polymerase chain reaction (PCR) arrays and real-time PCR, we also demonstrated that the methylation status of several critical tumor-suppressor genes increased as stage of breast disease increased, while miRNA-29b mRNA levels were significantly decreased in breast cancers versus normal breast. This increase in methylation status was accompanied by an increase in DNMT1 and DNMT3B mRNA in advanced stage of human breast cancers and in MCF-7, MDA-MB-361, HCC70, Hs-578T, and MDA-MB-231 breast cancer cells as compared to normal breast specimens and MCF-10-2A, a non-tumorigenic breast cell line, respectively. Our findings highlight the potential for a new epigenetic approach in improving breast cancer therapy by targeting DNMT3A and DNMT3B through miRNA-29b in non-invasive epithelial breast cancer cells.

Ethnic differences in DNA methyltransferases expression in patients with systemic lupus erythematosus.

PURPOSE: Systemic lupus erythematous (SLE) is a systemic autoimmune inflammatory disease with both genetic and epigenetic etiologies. Evidence suggests that deregulation of specific genes through epigenetic mechanisms may be a contributing factor to SLE pathology. There is increasing evidence that DNA methyltransferase activity may be involved. This study demonstrated modulation in expression of DNA methyltransferases (DNMTs) according to ethnicity in patients diagnosed with SLE. Furthermore, differential expression in one of the DNMTs was found in a subset of lupus patients on dehydroepiandrosterone (DHEA) therapy. METHODS: Real-time PCR analyses of DNMT1, DNMT3A and DNMT3B in peripheral blood mononuclear cells from a cohort of African American and European American lupus and non-lupus women were conducted. Also, global DNA methylation was assessed using the MethylFlash(TM) methylated quantification colorimetric assay. RESULTS: Significant increase in DNMT3A (p < 0.001) was shown in lupus patients when compared to age-matched healthy controls. This increase was associated with a higher SLEDI index. More striking was that expression levels for African American (AA) women were higher than European American women in the lupus populations. A subset of AA women on DHEA therapy showed a significant decrease (p < 0.05) in DNMT3A expression in comparison to lupus patients not on the therapy. DHEA is an androgenic steroid found in low levels in the serum of lupus patients. Supplementation of this hormone has been shown to be beneficial to some lupus patients. DHEA was not shown to effect DNMT1 or DNMT3B expression. Increased expression was also noted in DNMT3B (p < 0.05) in lupus patients compared to age-matched healthy controls. However, no significant difference was noted in DNMT1 (p = 0.2148) expression between lupus patients and healthy controls. Although increases were detected in de novo methyltransferases, a global decrease (p < 0.001) in 5-methycytosine was observed in lupus patients when compared to age-matched healthy controls. CONCLUSION: These findings suggest that epigenetic changes may play a critical role in the manifestations of the disease observed among ethnic groups, particularly African American women who often have a higher incidence of lupus. DHEA therapy effects on DNMT3A expression in AA women warrant further investigation in a larger population.

Cigarette smoke condensate induces differential expression and promoter methylation profiles of critical genes involved in lung cancer in NL-20 lung cells in vitro: short-term and chronic exposure.

Establishing early diagnostic markers of harm is critical for effective prevention programs and regulation of tobacco products. This study examined effects of cigarette smoke condensate (CSC) on expression and promoter methylation profile of critical genes (DAPK, ECAD, MGMT, and RASSF1A) involved in lung cancer development in different human lung cell lines. NL-20 cells were treated with 0.1-100 µg/ml of CSC for 24 to 72 hrs for short-term exposures. DAPK expression or methylation status was not significantly affected. However, CSC treatment resulted in changes in expression and promoter methylation profile of ECAD, MGMT, and RASSF1A. For chronic studies, cells were exposed to 1 or 10 µg/ml CSC up to 28 days. Cells showed morphological changes associated with transformation and changes in invasion capacities and global methylation status. This study provides critical data suggesting that epigenetic changes could serve as an early biomarker of harm due to exposure to cigarette smoke.

Characterization of UDP-glucuronosyltransferase (UGT1A1) Promoter Polymorphisms and Gene Expression on Ethnicity, Stage of Disease, and Menopausal Status in Breast Cancer.

Estrogen metabolism, catalyzed by UGTs, is a major drug-metabolic pathway that results in inactivation of estrogens and their metabolites. Alterations in UGTs involved in estrogen metabolism, has been suggested to play a role in breast cancer risk. The purpose of this study was to: 1) compare the mRNA expression levels of UGTs involved in estrogen metabolism in human breast tissues from women; 2) compare UGT1A1 mRNA expression to tumor stage, ethnicity, and menopausal status in a group of human breast tumors and normal breast tissues, and 3) investigate the association between variations in the number of TA repeats in the promoter region of UGT1A1 to gene expression. Quantification of UGT mRNA in breast tissues revealed that UGT1A4, UGT1A10, and UGT2B7 mRNA levels were decreased in breast cancers as compared to normal breast tissues. UGT1A1 mRNA levels were also significantly decreased in breast cancers as compared to normal breast tissues (Tumor: 0.5 ± 0.2; Normal: 4.1 ± 1.3, p = 0.0006). UGT1A1 mRNA down-regulation was strongly correlated with postmenopausal status in breast cancer versus controls (p = 0.04). In all the UGT1A1 genotypes observed in our study, the mean mRNA levels was significantly decreased among breast cancer cases as compared to controls for UGT1A1*1/*1 (p = 0.004), UGT1A1*28/*28 (p = 0.03) and UGT1A1*28/*37 (p = 0.06). Our findings demonstrate that further investigations are necessary to determine the role of UGT1A1 in breast carcinogenesis.

Enhanced efficacy of gemcitabine by indole-3-carbinol in pancreatic cell lines: the role of human equilibrative nucleoside transporter 1.

Pancreatic cancer patients treated with gemcitabine (2',2'-difluorodeoxycytidine) can eventually develop resistance. Recently, published data from our laboratory demonstrated enhanced efficacy of gemcitabine with the dietary agent, indole-3-carbinol (I3C). The current study examined the possible mechanism for this I3C-enhanced efficacy. Several pancreatic cell lines (BxPC-3, Mia Paca-2, PL-45, AsPC-1 and PANC-1) were examined for modulation of human equilibrative nucleoside transporter 1 (hENT1) expression, the major transporter for gemcitabine, by I3C alone and combined with gemcitabine. I3C significantly (p<0.01) up-regulated hENT1 expression in several cell lines. Gemcitabine alone showed no effect on hENT1 expression. However, combining gemcitabine with I3C further increased hENT1 expression. Cell viability assays revealed no effect of I3C on normal cells, hTERT-HPNE. hENT1-specific inhibitor, nitrobenzylthioinosine, significantly abrogated I3C-induced gemcitabine cytotoxicity, further demonstrating its specificity. This study demonstrates that up-regulation of hENT1 expression may be a novel mechanism involved in the additive effect of I3C and gemcitabine.

Transcriptional activity of DNMT3B in pancreatic cancer cells: effects of -149 (C→T) promoter polymorphism.

Polymorphic C-to-T change in the promoter region of DNA-methyltransferase-3B (DNMT3B) gene is associated with risk of several cancers. The aim of this study was to investigate the effect of DNMT3B promoter genetic variant on its transcriptional activity and to compare activity in several pancreatic cell lines. DNMT3B promoter constructs carrying either -149C allele or -149T allele were transiently transfected into pancreatic cancer cells. In promoter assaying, carriage of -149T allele showed only a slight activity (1.1-fold) in Mia cells (p=0.462). In contrast, significant increase (3.8-fold) in activity of -149T allele was shown in SU86.86 pancreatic cancer cells (p=0.0001). These preliminary findings suggest that genetic variance may influence DNMT3B expression in pancreatic cancer. Further studies are needed.

Food contaminant acrylamide increases expression of Cox-2 and nitric oxide synthase in breast epithelial cells.

Acrylamide has been discovered in foods cooked at high temperature. A potentially harmful effect of this dietary component has been suggested by data indicating its association with increased breast cancer. This study investigated the potential effects of acrylamide in nontumorigenic breast cells by assessing expression levels of inducible nitric oxide synthase (iNOS) and cycloogenase-2 (Cox-2) and NOS activity, which are known to be early molecular changes in disease formation. Treatment of cells with acrylamide increased levels of iNOS (both expression and activity) and Cox-2. Its potent metabolite, glycidamide, also induced both iNOS and Cox-2, with induction of iNOS occurring at a lower concentration. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), another food-borne carcinogen, was found to induce Cox-2 expression. Combining acrylamide with PhIP did not result in a further increase. These studies suggest that further research is needed to determine the role of carcinogens formed from cooking foods in inducing early molecular changes associated with breast cancer.