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

Comprehensive translational assessment of human-induced pluripotent stem cell derived cardiomyocytes for evaluating drug-induced arrhythmias

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) hold promise for assessment of drug-induced arrhythmias and are being considered for use under the comprehensive in vitro proarrhythmia assay (CiPA). We studied the effects of 26 drugs and 3 drug combinations on 2 commercially available iPSC-CM types using high-throughput voltage-sensitive dye and microelectrode-array assays being studied for the CiPA initiative and compared the results with clinical QT prolongation and torsade de pointes (TdP) risk. Concentration-dependent analysis comparing iPSC-CMs to clinical trial results demonstrated good correlation between drug-induced rate-corrected action potential duration and field potential duration (APDc and FPDc) prolongation and clinical trial QTc prolongation. Of 20 drugs studied that exhibit clinical QTc prolongation, 17 caused APDc prolongation (16 in Cor.4U and 13 in iCell cardiomyocytes) and 16 caused FPDc prolongation (16 in Cor.4U and 10 in iCell cardiomyocytes). Of 14 drugs that cause TdP, arrhythmias occurred with 10 drugs. Lack of arrhythmic beating in iPSC-CMs for the four remaining drugs could be due to differences in relative levels of expression of individual ion channels. iPSC-CMs responded consistently to human ether-a-go-go potassium channel blocking drugs (APD prolongation and arrhythmias) and calcium channel blocking drugs (APD shortening and prevention of arrhythmias), with a more variable response to late sodium current blocking drugs. Current results confirm the potential of iPSC-CMs for proarrhythmia prediction under CiPA, where iPSC-CM results would serve as a check to ion channel and in silico modeling prediction of proarrhythmic risk. A multi-site validation study is warranted

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

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. 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.sup.TM methylated quantification colorimetric assay. 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

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Adding 6 months of androgen deprivation therapy to postoperative radiotherapy for prostate cancer: a comparison of short-course versus no androgen deprivation therapy in the RADICALS-HD randomised controlled trial

Background Previous evidence indicates that adjuvant, short-course androgen deprivation therapy (ADT) improves metastasis-free survival when given with primary radiotherapy for intermediate-risk and high-risk localised prostate cancer. However, the value of ADT with postoperative radiotherapy after radical prostatectomy is unclear. Methods RADICALS-HD was an international randomised controlled trial to test the efficacy of ADT used in combination with postoperative radiotherapy for prostate cancer. Key eligibility criteria were indication for radiotherapy after radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to radiotherapy alone (no ADT) or radiotherapy with 6 months of ADT (short-course ADT), using monthly subcutaneous gonadotropin-releasing hormone analogue injections, daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as distant metastasis arising from prostate cancer or death from any cause. Standard survival analysis methods were used, accounting for randomisation stratification factors. The trial had 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 80% to 86% (hazard ratio [HR] 0·67). Analyses followed the intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov, NCT00541047. Findings Between Nov 22, 2007, and June 29, 2015, 1480 patients (median age 66 years [IQR 61–69]) were randomly assigned to receive no ADT (n=737) or short-course ADT (n=743) in addition to postoperative radiotherapy at 121 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 9·0 years (IQR 7·1–10·1), metastasis-free survival events were reported for 268 participants (142 in the no ADT group and 126 in the short-course ADT group; HR 0·886 [95% CI 0·688–1·140], p=0·35). 10-year metastasis-free survival was 79·2% (95% CI 75·4–82·5) in the no ADT group and 80·4% (76·6–83·6) in the short-course ADT group. Toxicity of grade 3 or higher was reported for 121 (17%) of 737 participants in the no ADT group and 100 (14%) of 743 in the short-course ADT group (p=0·15), with no treatment-related deaths. Interpretation Metastatic disease is uncommon following postoperative bed radiotherapy after radical prostatectomy. Adding 6 months of ADT to this radiotherapy did not improve metastasis-free survival compared with no ADT. These findings do not support the use of short-course ADT with postoperative radiotherapy in this patient population

Duration of androgen deprivation therapy with postoperative radiotherapy for prostate cancer: a comparison of long-course versus short-course androgen deprivation therapy in the RADICALS-HD randomised trial

Background Previous evidence supports androgen deprivation therapy (ADT) with primary radiotherapy as initial treatment for intermediate-risk and high-risk localised prostate cancer. However, the use and optimal duration of ADT with postoperative radiotherapy after radical prostatectomy remains uncertain. Methods RADICALS-HD was a randomised controlled trial of ADT duration within the RADICALS protocol. Here, we report on the comparison of short-course versus long-course ADT. Key eligibility criteria were indication for radiotherapy after previous radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to add 6 months of ADT (short-course ADT) or 24 months of ADT (long-course ADT) to radiotherapy, using subcutaneous gonadotrophin-releasing hormone analogue (monthly in the short-course ADT group and 3-monthly in the long-course ADT group), daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as metastasis arising from prostate cancer or death from any cause. The comparison had more than 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 75% to 81% (hazard ratio [HR] 0·72). Standard time-to-event analyses were used. Analyses followed intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov , NCT00541047 . Findings Between Jan 30, 2008, and July 7, 2015, 1523 patients (median age 65 years, IQR 60–69) were randomly assigned to receive short-course ADT (n=761) or long-course ADT (n=762) in addition to postoperative radiotherapy at 138 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 8·9 years (7·0–10·0), 313 metastasis-free survival events were reported overall (174 in the short-course ADT group and 139 in the long-course ADT group; HR 0·773 [95% CI 0·612–0·975]; p=0·029). 10-year metastasis-free survival was 71·9% (95% CI 67·6–75·7) in the short-course ADT group and 78·1% (74·2–81·5) in the long-course ADT group. Toxicity of grade 3 or higher was reported for 105 (14%) of 753 participants in the short-course ADT group and 142 (19%) of 757 participants in the long-course ADT group (p=0·025), with no treatment-related deaths. Interpretation Compared with adding 6 months of ADT, adding 24 months of ADT improved metastasis-free survival in people receiving postoperative radiotherapy. For individuals who can accept the additional duration of adverse effects, long-course ADT should be offered with postoperative radiotherapy. Funding Cancer Research UK, UK Research and Innovation (formerly Medical Research Council), and Canadian Cancer Society

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

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