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.

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.

Cystine-glutamate transporter SLC7A11 mediates resistance to geldanamycin but not to 17-(allylamino)-17-demethoxygeldanamycin.

The cystine-glutamate transporter SLC7A11 has been implicated in chemoresistance, by supplying cystine to the cell for glutathione maintenance. In the NCI-60 cell panel, SLC7A11 expression shows negative correlation with growth inhibitory potency of geldanamycin but not with its analog 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), which differs in the C-17 substituent in that the the methoxy moiety of geldanamycin is replaced by an amino group. Structure and potency analysis classified 18 geldanamycin analogs into two subgroups, "17-O/H" (C-17 methoxy or unsubstituted) and "17-N" (C-17 amino), showing distinct SLC7A11 correlation. We used three 17-O/H analogs and four 17-N analogs to test the role of the 17-substituents in susceptibility to SLC7A11-mediated resistance. In A549 cells, which are resistant to geldanamycin and strongly express SLC7A11, inhibition of SLC7A11 by (S)-4-carboxyphenylglycine or small interfering RNA increased sensitivity to 17-O/H, but had no effect on 17-N analogs. Ectopic expression of SLC7A11 in HepG2 cells, which are sensitive to geldanamycin and express low SLC7A11, confers resistance to geldanamycin, but not to 17-AAG. Antioxidant N-acetylcysteine, a precursor for glutathione synthesis, completely suppressed cytotoxic effects of 17-O/H but had no effect on 17-N analogs, whereas the prooxidant ascorbic acid had the opposite effect. Compared with 17-AAG, geldanamycin led to significantly more intracellular reactive oxygen species (ROS) production, which was quenched by addition of N-acetylcysteine. We conclude that SLC7A11 confers resistance selectively to 17-O/H (e.g., geldanamycin) but not to 17-N (e.g., 17-AAG) analogs partly as a result of differential dependence on ROS for cytotoxicity. Distinct mechanisms could significantly affect antitumor response and organ toxicity of these compounds in vivo.

Manganese superoxide dismutase-mediated gene expression in radiation-induced adaptive responses.

Antioxidant enzymes are critical in oxidative stress responses. Radioresistant variants isolated from MCF-7 human carcinoma cells following fractionated ionizing radiation (MCF+FIR cells) or overexpression of manganese superoxide dismutase (MCF+SOD cells) demonstrated dose-modifying factors at 10% isosurvival of 1.8 and 2.3, respectively. MCF+FIR and MCF-7 cells (exposed to single-dose radiation) demonstrated 5- to 10-fold increases in MnSOD activity, mRNA, and immunoreactive protein. Radioresistance in MCF+FIR and MCF+SOD cells was reduced following expression of antisense MnSOD. DNA microarray analysis and immunoblotting identified p21, Myc, 14-3-3 zeta, cyclin A, cyclin B1, and GADD153 as genes constitutively overexpressed (2- to 10-fold) in both MCF+FIR and MCF+SOD cells. Radiation-induced expression of these six genes was suppressed in fibroblasts from Sod2 knockout mice (-/-) as well as in MCF+FIR and MCF+SOD cells expressing antisense MnSOD. Inhibiting NF-kappa B transcriptional activity in MCF+FIR cells, by using mutant I kappa B alpha, inhibited radioresistance as well as reducing steady-state levels of MnSOD, 14-3-3 zeta, GADD153, cyclin A, and cyclin B1 mRNA. In contrast, mutant I kappa B alpha was unable to inhibit radioresistance or reduce 14-3-3 zeta, GADD153, cyclin A, and cyclin B1 mRNAs in MCF+SOD cells, where MnSOD overexpression was independent of NF-kappa B. These results support the hypothesis that NF-kappa B is capable of regulating the expression of MnSOD, which in turn is capable of increasing the expression of genes that participate in radiation-induced adaptive responses.

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.

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.

Increased expression of heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP) in pancreatic tissue from smokers and pancreatic tumor cells.

Pancreatic cancer is a major cause of deaths in the United States, and has one of the lowest 5-year survival rates. Early diagnosis has not been possible due to the lack of reliable early tumor markers. The heterogeneous nuclear ribonucleoprotein A1/B2 (hnRNP) was recently shown to be up-regulated in the early stage of lung cancer. This protein plays an important role in biogenesis and transport of mRNA. Up-regulation of hnRNP usually precedes morphological differentiation and is considered a good biomarker in the early stages of cancer development. Because smoking is a high risk factor for pancreatic cancer, this study examined the expression of hnRNP in human pancreatic tissues from smokers and non-smokers. A two-fold increase in expression of hnRNP was found overall in smokers when compared to non-smokers and smokers who quit (P<0.05). The increase in expression of hnRNP was higher in female smokers compared to female non-smokers. High levels of expression was also shown in a limited number of human pancreatic adenocarcinomas and two pancreatic tumor cell lines, HPAF-11 and SU 86.86. HP-8, a normal primary pancreatic cell line, did not express hnRNP. These results strongly suggest that up-regulation of hnRNP may be a good candidate for early screening for pancreatic cancer because of its activation in pancreatic tissue from smokers and activation in pancreatic adenocarcinomas. Over-expression of hnRNP has been suggested as evidence that normal transcriptional regulation is altered.

Modulation of the constitutive activated STAT3 transcription factor in pancreatic cancer prevention: effects of indole-3-carbinol (I3C) and genistein.

BACKGROUND: The signal transducer and activator of transcription 3 (STAT3) is a latent transcription factor required in proliferation and differentiation. STAT3 is activated constitutively in a number of cancers. MATERIALS AND METHODS: This study was conducted to assess the possible involvement of STAT3 activation in pancreatic cancer and the potential for this pathway as a target in chemopreventive strategy. RESULTS: STAT3 was shown for the first time to be constitutively activated in human pancreatic carcinoma specimens but not in normal pancreatic tissues. Constitutively activated STAT3 was also found in pancreatic tumor cell lines (Panc-1 and MIA PaCa-2) which could be modulated by indole-3-carbinol (13C) and genistein. At concentrations higher than 10 microM, STAT3 constitutive activation is inhibited by both agents. Induction of apoptosis by 13C was also demonstrated. CONCLUSION: Given its critical role in tumorigenesis, our results suggest that STAT3 activation provides an important and appropriate target for chemoprevention in pancreatic cancer treatment.

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.

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.

Gender differences in gemcitabine (Gemzar) efficacy in cancer cells: effect of indole-3-carbinol.

Pancreatic cancer has a poor prognosis, mainly due to lack of effective therapies. This study demonstrated the ability of dietary agent, indole-3-carbinol (I3C), to lower the LD(50) of gemcitabine (Gemzar) in decreasing growth of both male (MiaPaca2) and female (SU86.86) pancreatic cancer cells. Female pancreatic cancer cells were more resistant to gemcitabine alone. Additionally, RT-PCR analysis of MiaPaca2 cells treated with 1, 10 or 100 µM of I3C showed that I3C reactivated the tumor suppressor gene p16INK4a in pancreatic cancer cells. Methylated-specific PCR analysis indicated that I3C demethylated the promoter region of p16 INK4a, which was methylated in the untreated cancer cells. p16INK4a inactivation through promoter hypermethylation is considered an early event in pancreatic carcinogenesis. A positive control using 5-azacytidine also reactivated p16INK4a. This study demonstrated the potential of I3C, a possible non-toxic hypomethylating agent, combined with the anticancer agent, gemcitabine, to be a powerful strategy for treating pancreatic cancer.

Acrylamide decreased dopamine levels and increased 3-nitrotyrosine (3-NT) levels in PC 12 cells.

Acrylamide is a chemical known to produce neurotoxicity in animals, as well as in humans. The mechanism of acrylamide-induced neurotoxicity is not fully known. However, recent studies have revealed that acrylamide affects the dopaminergic system. Therefore, the aim of this study was to investigate the effect of acrylamide on dopamine (DA) and the metabolites, 3,4-dihydroxy phenylacetic acid (DOPAC) and homovanillicacid (HVA), levels in Pheochromocytoma (PC 12) cells. In addition, the generation of peroxynitrite (ONOO(-)), measured by 3-nitrotyrosine (3-NT), was investigated as a possible mechanism in acrylamide-induced neurotoxicity. HPLC-coupled to electrochemical detection (ECD) was used to determine DA, DOPAC, HVA and 3-NT levels. Acrylamide (0.01-5mM) exposure produced a dose- and time (1-42h)-dependent decrease in DA levels. The decrease (P<0.05) in DA levels was noted at 24h after exposure to acrylamide. The study also revealed that 3-NT levels in PC 12 increased as a result of treatment with acrylamide. Thus, these data suggest that acrylamide-induced decrease in DA levels in PC 12 cells may be associated with peroxynitrite formation, measured as 3-NT levels.

Age and gender affect DNMT3a and DNMT3b expression in human liver.

DNA methylation is catalyzed by a family of DNA methyltransferases (DNMTs) including the maintenance enzyme DNMT 1 and de novo methyltransferases DNMT 3a and DNMT 3b. Elevated levels of DNMTs have been found in cancer cells and in several types of human tumors. A polymorphism found in DNMT3b has been associated with increased risk for several cancers. The factors influencing DNMT expression in human tissues have not been clearly determined. he present study examined TDNMT3a and DNMT3b levels in human liver tissue samples and compared the effect of ageing, cigarette smoking, and gender. DNMT3a and DNMT3b expression levels in the samples from older individuals (56-78 years, n = 28) were both significantly higher than those of the younger group (16-48 years, n = 27) (73.2 +/- 3.4 vs 8.3 +/- 2.8 and 56.1 +/- 1.9 vs 17.5 +/- 5.7, respectively; p < 0.05). Levels of DNMT3b in females were significantly higher than those in males (75.4 +/- 2.2 vs 16.3 +/- 4.7; p < 0.05); however, DNMT3a levels were similar for females and males (52.7 +/- 2.7 vs 48.4 +/- 2.0). Expression levels of DNMT3a and DNMT3b were similar in smokers and nonsmokers (58.1 +/- 3.5 vs 60.8 +/- 3.1 and 54.5 +/- 2.3 vs 48.3 +/- 1.8, respectively). Genotyping for DNMT3b (C-->T) variant in this sample pool showed a frequency distribution of CC (41%), CT (50%), and TT (9%). The findings from this study suggest that ageing and gender may be important factors influencing DNA methylation status.

Search for an association between the human CYP1A2 genotype and CYP1A2 metabolic phenotype.

The genotype responsible for more than 60-fold interindividual differences in human hepatic CYP1A2 constitutive expression is not understood. Resequencing the human CYP1A1_CYP1A2 locus (39.6 kb) in five major geographically isolated subgroups recently led to the identification of 85 single nucleotide polymorphisms (SNPs), 57 of which were double-hit SNPs. Here, we attempted to correlate the CYP1A2 genotype with a metabolic phenotype. We chose 16 SNPs (all having a minor allele frequency > or =0.05 in Caucasians) to genotype 32 DNA samples (26 Caucasians, six Ethiopians) in which CYP1A2 metabolism had previously been determined. From 280 subjects (five locations worldwide) that had been CYP1A2-phenotyped, we genotyped the 10 highest, 14 lowest and eight intermediate DNA samples. Although no SNP was significant (P<0.05), possibly due to the small sample size, we found a trend for several of the six SNPs across the CYP1A2 linkage disequilibrium block associated with the trait. Five CYP1A2 haplotypes were inferred, two of which had not previously been reported; haplotype 1A2H10 showed the greatest association with CYP1A2 activity. Regulatory sequences responsible for the large interindividual differences in hepatic CYP1A2 gene basal expression might reside, in part, with some of these CYP1A2 SNPS but, in large part, might be located either cis (in nearby sequences not yet haplotyped) or trans in that they are not linked to the gene. We conclude that no SNP or haplotype in the CYP1A2 gene has yet been identified that can unequivocally be used to predict the metabolic phenotype in any individual patient.

Characterization of a common deletion polymorphism of the UGT2B17 gene linked to UGT2B15.

Members of the human UDP-glucuronosyltransferase 2B family are located in a cluster on chromosome 4q13 and code for enzymes whose gene products are responsible for the normal catabolism of steroid hormones. Two members of this family, UGT2B15 and UGT2B17, share over 95% sequence identity. However, UGT2B17 exhibits broader substrate specificity due to a single amino acid difference. Using gene-specific primers to explore the genomic organization of these two genes, it was determined that UGT2B17 is absent in some human DNA samples. The gene-specific primers demonstrated the presence or absence of a 150 kb genomic interval spanning the entire UGT2B17 gene, revealing that UGT2B17 is present in the human genome as a deletion polymorphism linked to UGT2B15. Furthermore, it is shown that the UGT2B17 deletion polymorphism shows Mendelian segregation and allele frequencies that differ between African Americans and Caucasians.