Multidrug-related protein 1 (MRP1) polymorphisms rs129081, rs212090, and rs212091 predict survival in normal karyotype acute myeloid leukemia.

Resistant disease is still a main obstacle in acute myeloid leukemia (AML) treatment. Therefore, individual genetic variations affecting therapy response are gaining increasing importance. Both SNPs and ABC transporter genes could already be associated with drug resistance. Here, we report allelic variants of MRP1 (ABCC1) SNPs rs129081, rs212090, and rs212091 with significant influences on survival in AML patients. DNA was extracted from bone marrow samples (n = 160) at diagnosis. Genotyping 48 SNPs within seven different ABC transporter genes using real-time PCR revealed rs129081 GG variant with a significant higher OS (p = 0.035) and DFS (p = 0.01). Comparing TT and AA rs212090 variants showed significant influences on DFS (p = 0.021). SNP rs212091 GG expression was associated with worse OS (p = 0.006) and a significant difference in DFS between alleles GG and AA (p = 0.018). The multivariable models confirmed a significant influence on OS for rs212091 (AA HR = 0.296, 95% CI 0.113-0.774, p = 0.013 and GG p = 0.044). Rs129081 variant CG, TT of rs212090, AA, and AG of rs212091 demonstrated significant impact on DFS (p = 0.024, p = 0.029, p = 0.017, and p = 0.042, respectively). This analysis demonstrates a significant influence of MRP1 SNPs on survival in AML. As they were not associated to prognostic characteristics, we suggest these SNPs to be independent prognostic markers for AML.

Trastuzumab in Esophagogastric Cancer: HER2-Testing and Treatment Reality outside Clinical Studies in Germany.

We analysed trends over time in palliative first-line chemotherapy in patients with locally advanced or metastatic esophagogastric cancer. Special focus was on frequency and quality of HER2-testing and trends in drug use in combination with trastuzumab. Earlier published data about patients treated outside clinical studies showed a relatively low rate of HER2-testing and insufficient test quality. A total of 2,808 patients retrospectively documented in Therapiemonitor (®) from 2006 to 2013 were analysed regarding treatment intensity and trends in used drugs. Data on HER2-testing and therapies were analysed in two cohorts documented in 2010 and 2011 (1) compared to 2012 and 2013 (2). Treatment intensity increased: 49.3% of patients received at least a triplet in 2013 compared to 10.1% in 2006. In cohort 2 HER2 expression was tested in 79.1% of the cases. Still, in 26.9% testing was not done as requested by guidelines. Good performance status, multiple metastases, age ≤ 65 years, the objective "to prevent progression," good cognitive capabilities, estimated good compliance, and social integration positively influenced the probability of HER2-testing; comorbidities negatively affected it. Usage of the combination of fluoropyrimidines and cisplatin with trastuzumab declined from 67% in cohort 1 to 50% in cohort 2.

Estrogen Receptor Alpha (ESR1) Single-Nucleotide Polymorphisms (SNPs) Affect Malignant Melanoma Susceptibility and Disease Course.

The incidence of malignant melanoma in the developed world is continuously increasing. We conducted a case-control study in order to evaluate the association between each of the four estrogen receptor alpha polymorphisms (ESR1 single-nucleotide polymorphisms [SNPs] +2464C/T, -4576A/C, +1619A/G, and +6362C/T) and malignant melanoma susceptibility and disease course. The study population consisted of 205 Caucasian patients who were diagnosed as having malignant melanoma and 208 healthy Caucasian controls. Through DNA genotyping, we identified a SNP-dependent malignant melanoma susceptibility as well as a SNP-dependent effect on the course of disease and response to therapy.

Cooking enhances curcumin anti-cancerogenic activity through pyrolytic formation of "deketene curcumin".

Curcumin is widely used in traditional Asian kitchen as a cooking ingredient. Despite its low bioavailability, epidemiological data, on low cancer incidence in Asia, suggest beneficial health effects of this compound. Therefore, the question arose whether cooking modifies the anti-cancerogenic effects of curcumin. To evaluate this, we pyrolysed curcumin with and without coconut fat or olive oil, and analysed the products by high-performance liquid chromatography (HPLC). A number of more hydrophilic curcumin isoforms and decomposition products, including a compound later identified by nuclear magnetic resonance spectroscopy (NMR) as "deketene curcumin" (1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadiene-3-one), formerly described as a synthetic curcumin derivative, were detected. Additionally, we proved that deketene curcumin, compared to curcumin, exhibits higher toxicity on B78H1 melanoma cells resulting in G2 arrest. In conclusion, deketene curcumin is formed as a consequence of pyrolysis during common household cooking, showing stronger anti-cancer effects than curcumin. Moreover, we propose a chemical reaction-pathway for this process.

GRHL1 acts as tumor suppressor in neuroblastoma and is negatively regulated by MYCN and HDAC3.

Neuroblastoma is an embryonic solid tumor of neural crest origin and accounts for 11% of all cancer-related deaths in children. Novel therapeutic strategies are therefore urgently required. MYCN oncogene amplification, which occurs in 20% of neuroblastomas, is a hallmark of high risk. Here, we aimed to exploit molecular mechanisms that can be pharmacologically addressed with epigenetically modifying drugs, such as histone deacetylase (HDAC) inhibitors. Grainyhead-like 1 (GRHL1), a gene critical for Drosophila neural development, belonged to the genes most strongly responding to HDAC inhibitor treatment of neuroblastoma cells in a genome-wide screen. An increase in the histone H4 pan-acetylation associated with its promoter preceded transcriptional activation. Physically adjacent, HDAC3 and MYCN colocalized to the GRHL1 promoter and repressed its transcription. High-level GRHL1 expression in primary neuroblastomas correlated on transcriptional and translational levels with favorable patient survival and established clinical and molecular markers for favorable tumor biology, including lack of MYCN amplification. Enforced GRHL1 expression in MYCN-amplified neuroblastoma cells with low endogenous GRHL1 levels abrogated anchorage-independent colony formation, inhibited proliferation, and retarded xenograft growth in mice. GRHL1 knockdown in MYCN single-copy cells with high endogenous GRHL1 levels promoted colony formation. GRHL1 regulated 170 genes genome-wide, and most were involved in pathways regulated during neuroblastomagenesis, including nervous system development, proliferation, cell-cell adhesion, cell spreading, and cellular differentiation. In summary, the data presented here indicate a significant role of HDAC3 in the MYCN-mediated repression of GRHL1 and suggest drugs that block HDAC3 activity and suppress MYCN expression as promising candidates for novel treatment strategies of high-risk neuroblastoma.

Curcumin intake affects miRNA signature in murine melanoma with mmu-miR-205-5p most significantly altered.

Melanoma is the most aggressive form of skin cancer with estimated 48,000 deaths per year worldwide. The polyphenol curcumin derived from the plant Curcuma longa is well known for its anti-inflammatory and anti-cancerogenic properties. Accordingly, dietary intake of this compound may be suitable for melanoma prevention. However, how this compound affects basic cellular mechanisms in developing melanoma still remains elusive. Therefore, the aim of this study was to investigate for the first time the impact of oral curcumin administration on the miRNA signature of engrafting melanoma. For this purpose, the effects of a 4% curcumin diet were tested on melanoma, which were established by injection of murine B78H1 cells in the flank of C57BL/6 mice. Curcumin diet or standard chow (control) was administered two weeks prior to injection of tumor cells until termination of the experiment. High throughput chip-based array analysis was deployed to detect alterations in the miRNA signature of the tumors. Curcumin treatment significantly reduced the growth of the flank tumors. Furthermore the miRNA expression signature in tumors was substantially altered by curcumin intake with mmu-miR-205-5p over 100 times higher expressed when compared to controls. The expression levels of identified key miRNAs in the tumor samples were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). A comparable expression pattern of these miRNAs was also detected in other curcumin-treated melanoma cell lines under in vitro conditions. Putative targets of curcumin-induced up-regulated miRNAs were enriched in 'o-glycan biosynthesis', 'endoplasmatic reticulum protein processing' and different cancer-related pathways. Western Blot analyses revealed that of these targets anti-apoptotic B-cell CLL/lymphoma 2 (Bcl-2) and proliferating cell nuclear antigen (PCNA) were significantly down-regulated in curcumin-treated tumors. These findings demonstrate a profound alteration of the miRNA expression signature in engrafting curcumin-treated melanoma with mmu-miR-205-5p being up-regulated most significantly.

Transcription factor NFκB regulates the expression of the histone deacetylase SIRT1.

BACKGROUND: The NAD-dependent protein deacetylase SIRT1 has a wide range of different targets, which may be regulated either directly through deacetylation and thus potentially altering their activity or localization or indirectly by deacetylation of histones, which in turn alters their transcription rate and availability. SIRT1 is therefore involved in the regulation of many different and fundamental cellular processes such as apoptosis, metabolism, differentiation and cell cycle arrest. It is also involved in the regulation of resistance of cells against oxidative stress and longevity under conditions of caloric restriction. Even though the targets and role of SIRT1 have been studied quite intensively, only little is known about the mechanisms affecting SIRT1 transcriptional regulation. The nuclear factor NFκB is a well-studied and widely known transcription factor, which is involved in the regulation of many important cellular activities. The regulation of NFκB by SIRT1 has been reported recently, but it is, however, still unknown whether a feedback mechanism affects the regulation of SIRT1 too, particularly in view of the fact that putative NFκB binding sites within the SIRT1 promoter suggest just that. RESULTS: In the study presented herein we show that there is activation of the SIRT1 promoter by overexpression of different NFκB subunits. Direct binding of NFκB to the SIRT1 promoter can be demonstrated by an electrophoretic mobility shift assay. Further investigations indicated enhanced expression of SIRT1 on the mRNA levels in cells overexpressing NFκB. A functional assay showed that acetylation of one of the main target proteins of SIRT1 is reduced in these cells. CONCLUSIONS: These finding together indicate SIRT1 expression to be regulated in a positive feedback loop by NFκB. The putative binding sites for NFκB found within the SIRT1 promoter appears to be functional and several NFκB subunits are able to enhance the expression of SIRT1 if they are overexpressed.

ESR1 single nucleotide polymorphisms predict breast cancer susceptibility in the central European Caucasian population.

Estrogen and progesterone hormones are key regulators of a wide variety of biological processes. In addition to their influence on reproduction, cell differentiation and apoptosis, they affect inflammatory response, cell metabolism and most importantly, they regulate physiological breast tissue proliferation and differentiation as well as the development and progression of breast cancer. In order to assess whether genetic variants in the steroid hormone receptor gene ESR1 (estrogen receptor alpha) had an effect on sporadic breast cancer susceptibility, we assessed 7 ESR1 single nucleotide polymorphisms (SNPs) for associations with breast cancer susceptibility and clinical parameters in 221 breast cancer patients and 221 controls, respectively. We identified ESR1 intron SNP +2464 C/T (rs3020314) and ESR1 intron SNP -4576 A/C (rs1514348) to correlate with breast cancer susceptibility and progesterone receptor expression status. Patients genotyped CT for ESR1 intron SNP +2464 (rs3020314) (p ≤ 0.045) or genotyped AC for ESR1 intron SNP -4576 (rs1514348) (p ≤ 0.000026) were identified to carry a significant risk as to the development of breast cancer in the Central European Caucasian population (both together: p ≤ 0.000488). Our study could confirm previous associations and revealed new associations of SNP rs1514348 with susceptibility to breast cancer and clinical outcome, which might be used as new additional SNP markers.

Cloning, chromosomal characterization and FISH mapping of the NAD(+)-dependent histone deacetylase gene sirtuin 5 in the mouse.

Sirtuin 5 (SIRT5) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, belonging to the silent information regulator 2 (Sir2) family of sirtuin histone deacetylases (sirtuins). The yeast Sir2 protein and its mammalian derivatives are important in epigenetic gene silencing, DNA repair and recombination, cell cycle, microtubule organization and in the regulation of aging. In mammals, 7 sirtuin isoforms have been identified to date of which three (SIRT3, SIRT4 and SIRT5) are localized in the mitochondria, which serve as the center of energy management and the initiation of cellular apoptosis. In the study presented herein, we report the genomic organization and chromosomal localization of the murine sirt5 gene. We have isolated and characterized the murine sirt5 genomic sequence, which spans a region of 24,449 bp and which has one single genomic locus. The murine sirt5 gene consists of 8 exons and encodes a 310-aa protein with a predictive mo-lecular weight of 34.1 kDa and an isoelectric point of 8.90. For the murine sirt5 gene only one single genomic locus has been identified. The gene has been localized to mouse chromosome 13A4 and is flanked by STS-marker 164522 (synonymous WI MRC-RH: 506859).

DNMT1 genetic polymorphisms affect breast cancer risk in the central European Caucasian population.

INTRODUCTION: DNA methylation of CpG islands within the promoter region of genes is an epigenetic modification with an important role in the development of cancer and it is typically mediated by DNA methyltransferases (DNMTs). In cancer cells, global hypomethylation of the genome as a whole and regional hypermethylation of CpG islands have been reported. Four groups of DNMTs have been identified: DNMT1, DNMT2 (TRDMT1), DNMT3A and DNMT3B. DNMT2 uses the catalytic mechanism of DNMTs, but does in fact methylate RNA. Little is known about the significance of these genes in human breast cancer. In the study presented herein, we analyzed five distinct DNMT single SNPs with regard to potential associations with breast cancer risk. CASE DESCRIPTION: In this study, we genotyped 221 female Caucasian breast cancer patients and 221 female Caucasian healthy controls, and we used five allele-specific real-time polymerase chain reaction (qPCR) assays. We selected one locus within the DNMT1 gene and two loci within the DNMT3A and DNMT3B genes, respectively. Statistics were calculated using the chi-squared and Fisher's exact tests, and correlated with clinical parameters such as age, diagnosis, histology, TNM stage, hormonal receptor status, human epidermal growth factor receptor 2 (HER2) status, response to treatment and survival. Statistically significant results were obtained for correlations with the DNMT1 gene. DISCUSSION AND EVALUATION: Five genomic loci within the DNMT1, DNMT3A and DNMT3B genes were assessed. Statistical significance (P = 0.030) was identified for DNMT1 SNP (A201G, rs2228612): six women within the control group were GG homozygous (variant), while this mutation was absent in the breast cancer group. CONCLUSIONS: We conclude that women with the DNMT1 SNP (A201G, rs2228612) GG homozygous genotype (variant) have a lower risk of developing breast cancer compared to heterozygous or wildtype genotypes. To date, alterations within the DNMT1 gene have not been reported to be associated with cancer in the Caucasian population.

Sirtuin1 single nucleotide polymorphism (A2191G) is a diagnostic marker for vibration-induced white finger disease.

BACKGROUND: Vibration-induced white finger disease (VWF), also known as hand-arm vibration syndrome, is a secondary form of Raynaud's disease, affecting the blood vessels and nerves. So far, little is known about the pathogenesisof the disease. VWF is associated with an episodic reduction in peripheral blood flow. Sirtuin 1, a class III histone deacetylase, has been described to regulate the endothelium dependent vasodilation by targeting endothelial nitric oxide synthase. We assessed Sirt1single nucleotide polymorphisms in patients with VWF to further elucidate the role of sirtuin 1 in the pathogenesis of VWF. METHODS: Peripheral blood samples were obtained from 74 patients with VWF (male 93.2%, female 6.8%, median age 53 years) and from 317 healthy volunteers (gender equally distributed, below 30 years of age). Genomic DNA was extracted from peripheral blood mononuclear cells and screened for potential Sirt1single nucleotide polymorphisms. Four putative genetic polymorphisms out of 113 within the Sirt1 genomic region (NCBI Gene Reference: NM_012238.3) were assessed. Allelic discrimination was performed by TaqMan-polymerasechainreaction-based allele-specific genotyping single nucleotide polymorphism assays. RESULTS: Sirt1single nucleotide polymorphism A2191G (Assay C_25611590_10, rs35224060) was identified within Sirt1 exon 9 (amino acid position 731, Ile → Val), with differing allelic frequencies in the VWF population (A/A: 70.5%, A/G: 29.5%, G/G: 0%) and the control population (A/A: 99.7%, A/G: 0.3%, G/G: 0.5%), with significance levels of P < 0.001 (Mann-Whitney U test (two-tailed) P <0.001; F-exact t-test and Chi-square test with Yates correction (all two-tailed): P <0.0001). The heterogeneous A/G genotype in base pair position 2191 is significantly overrepresented in the VWF patient population when compared with healthy controls. CONCLUSION: We identified theSirt1A2191Gsingle nucleotide polymorphism as a diagnostic marker for VWF.

Therapeutic platelet transfusion versus routine prophylactic transfusion in patients with haematological malignancies: an open-label, multicentre, randomised study.

BACKGROUND: Routine prophylactic platelet transfusion is the standard of care for patients with severe thrombocytopenia. We assessed the effect of a new strategy of therapeutic platelet transfusion on the number of transfusions and safety in patients with hypoproliferative thrombocytopenia. METHODS: We did a multicentre, open-label, randomised parallel-group trial at eight haematology centres in Germany. Patients aged 16-80 years, who were undergoing intensive chemotherapy for acute myeloid leukaemia or autologous haemopoietic stem-cell transplantation for haematological cancers, were randomly assigned via a computer-generated randomisation sequence to receive either platelet transfusion when bleeding occurred (therapeutic strategy) or when morning platelet counts were 10×10(9) per L or lower (prophylactic strategy). Investigators undertaking interventions were not masked to group assignment. The primary endpoint was the number of platelet transfusions. Analysis was by intention to treat. This trial is registered, NCT00521664. FINDINGS: 197 patients were assigned the prophylactic strategy and 199 the therapeutic strategy. Of 391 patients analysed, the therapeutic strategy reduced the mean number of platelet transfusions by 33·5% (95% CI 22·2-43·1; p<0·0001) in all patients (2·44 [2·22-2·67] in prophylactic group vs 1·63 [1·42-1·83] in therapeutic group), 31·6% (18·6-42·6; p<0·0001) in those with acute myeloid leukaemia (2·68 [2·35-3·01] vs 1·83 [1·58-2·10]), and 34·2% (6·6-53·7; p=0·0193) in those who had had autologous transplantation (1·80 [1·45-2·15] vs 1·18 [0·82-1·55]. We noted no increased risk of major haemorrhage in patients who had undergone autologous transplantation. In those with acute myeloid leukaemia, risk of non-fatal grade 4 (mostly CNS) bleeding was increased. We recorded 15 cases of non-fatal haemorrhage: four retinal in each transfusion group, and one vaginal and six cerebral in the therapeutic group. 12 patients died in the study: two from fatal cerebral haemorrhages in the therapeutic group, and ten (five in each treatment group) unrelated to major bleeding. INTERPRETATION: The therapeutic strategy could become a new standard of care after autologous stem-cell transplantation; however, prophylactic platelet transfusion should remain the standard for patients with acute myeloid leukaemia. The new strategy should be used by some haematology centres only if the staff are well educated and experienced in the new approach and can react in a timely way to first signs of CNS bleeding. FUNDING: Deutsche Krebshilfe eV (German Cancer Aid).

On the epigenetics of vascular regulation and disease.

Consolidated knowledge is accumulating as to the role of epigenetic regulatory mechanisms in the physiology of vascular development and vascular tone as well as in the pathogenesis of cardiovascular disease. The modulation of gene expression through modification of the epigenome by structural changes of the chromatin architecture without alterations of the associated genomic DNA sequence is part of the cellular response to environmental changes. Such environmental conditions, which are finally being translated into adaptations of the cardiovascular system, also comprise pathological conditions such as atherosclerosis or myocardial infarction. This review summarizes recent findings on the epigenetics of vascular regulation and disease and presents nutritional and pharmacological approaches as novel epigenetic strategies in the prevention and treatment of cardiovascular disease.

Involvement of sirtuins in life-span and aging related diseases.

Since healthy aging remains one of the ideals of modern society, both, the identification of the underlying molecular mechanisms and interventions regarding the aging process are of considerable interest. Among the mechanisms currently being considered, the sirtuin family of histone deacetylases have been implicated to play a crucial role during the aging process both due to their requirement of NAD(+) as a cofactor for enzymatic activity, which determines a crucial link between sirtuins and the energy dependent regulation of gene transcription and their versatile target substrates mainly consisting of key regulators of metabolic, stress and cell cycle control. This chapter summarizes current evidences linking sirtuins to aging and outlines their potential as promising therapeutic targets for the treatment of age-related diseases.

Signature of VDR miRNAs and epigenetic modulation of vitamin D signaling in melanoma cell lines.

BACKGROUND: Melanoma cells express the nuclear vitamin D receptor (VDR), indicating that malignant melanoma represents a promising target for treatment with 1,25-dihydroxyvitamin D3 (1,25(OH)(2)D(3)) or its analogs. We previously showed that some melanoma cell lines are resistant to the antiproliferative effects of 1,25(OH)(2)D(3) and that 1,25(OH)(2)D(3)-sensitivity can, at least in part, be restored by co-treatment with the histone deacetylase inhibitor (HDACI) Trichostatin A (TSA) or with the DNA methyltransferase inhibitor (DNMTI), 5-azacytidine (5-Aza). This study aimed at gaining further insights into the molecular mechanisms that underlie the epigenetic modulation of 1,25(OH)(2)D(3)-sensitivity in melanoma cells. MATERIALS AND METHODS: The expression of VDR mRNA, protein and two candidates of VDR microRNAs (miR-125b, miR-27b) were compared in 1,25(OH)(2)D(3)-responsive (MeWo, SK-Mel28) and -resistant (SK-Mel5, IGR) melanoma cell lines and in normal human melanocytes (NHM) using real time PCR and western blot analysis. Additionally, the effect of 1,25(OH)(2)D(3), epigenetic modulating drugs (TSA, 5-Aza) and miR-125b antisense on the expression of VDR messenger RNA (mRNA)/protein, miR-125b and miR-27b was investigated. RESULTS: Treatment with 1,25(OH)(2)D(3) and/or epigenetic drugs (5-Aza, TSA) modulated the VDR mRNA expression in the 1,25(OH)(2)D(3)-responsive and - resistant melanoma cell lines and in the NHM. Treatment with 5-Aza, but not with TSA, reduced the expression of miR-125b in the 1,25(OH)(2)D(3)-responsive and -resistant melanoma cell lines and in the NHM. Treatment with 1,25(OH)(2)D(3) and/or epigenetic drugs (5-Aza, TSA) reduced the miR-27b expression in three out of four melanoma cell lines. Moreover, no difference was observed in VDR protein expression in the 1,25(OH)(2)D(3)-responsive as compared to the 1,25(OH)(2)D(3)-resistant melanoma cell lines. Transfection with miR-125b antisense did not affect the VDR mRNA/protein expression in the IGR cells. CONCLUSION: Responsiveness to 1,25(OH)(2)D(3) corresponds to the expression level of VDR mRNA which in turn might be regulated by VDR microRNAs or epigenetic modulating drugs.

The T_T genotype within the NME1 promoter single nucleotide polymorphism -835 C/T is associated with an increased risk of cytarabine induced neurotoxicity in patients with acute myeloid leukemia.

Recently, numerous studies have been published on inter-individual variations in the response to specific treatment with cytostatic agents such as cytarabine (Ara-C) in patients with acute myeloid leukemia (AML). Differences at the genetic level and potentially associated changes in the expression and/or function of specific drug metabolizing enzymes appear to play an important role in this inter-individual susceptibility. Single nucleotide polymorphisms (SNPs) can be easily assessed in order to further investigate and explain inter-individual differences as to Ara-C associated toxicity and response to treatment. In this retrospective study we correlated five SNPs within the NME1 promoter with drug-induced toxicity, disease-free survival and overall survival (OS) in 360 Caucasian patients suffering from AML. A significant correlation between SNPs and disease-free survival or overall survival was not found. For the NME1 promoter SNP - 835 C/T (rs2302254) we identified a significant correlation between low platelet counts and better Eastern Cooperative Oncology Group performance status (grade 3/4). An increased risk of neurotoxicity was identified for the NME1 promoter SNP - 835 C/T (rs2302254) genotype T_T. Multivariate analyses also showed that these variables were independent risk factors. Ara-C causes neuronal cell death by introduction of apoptosis with reactive oxygen species, causing oxidative DNA damage and initiating the p53-dependent apoptotic program. Recent data show that oral administration of the antioxidant N-acetylcysteine for 14 days is able to prevent Ara-C induced behavioral deficits and cellular alterations of the adult cerebellum in a rat model.

Epigenetic regulation of cellular adhesion in cancer.

Epigenetics describes the development and maintenance of stable heritable gene expression patterns, which allow cells to show different phenotypes despite of a commonly shared genetic code. The increasing knowledge in this field during the last decades reveals its importance for many physiological processes like differentiation, embryogenesis and parental imprinting, but also for some diseases such as cancer. Recent data have shown that the complexity of carcinogenesis can no longer be explained solely on the basis of genetic changes, but epigenomic alterations such as changes of the DNA methylation pattern and/or post-translational histone modifications and changes of microRNA expression need to be equally considered. Such epigenetic alterations may cause permanent changes in gene expression patterns and may therefore essentially contribute to some of the known phenotypic characteristics of cancer cells like the loss of growth control, altered intercellular communication and enhanced motility. The two latter may essentially be associated with the downregulation of cellular adhesion molecules, which may therefore be relevant in the context of cancer invasiveness and prognosis. The targeted modification of the epigenome may therefore open new horizons within the increasingly important field of epigenetic therapeutics-particularly in view of the regulation of cellular adhesion with particular attention to tumor cell invasion and metastasis.

Ribavirin restores ESR1 gene expression and tamoxifen sensitivity in ESR1 negative breast cancer cell lines.

Tumor growth is estrogen independent in approximately one-third of all breast cancers, which makes these patients unresponsive to hormonal treatment. This unresponsiveness to hormonal treatment may be explained through the absence of the estrogen receptor alpha (ESR1). The ESR1 gene re-expression through epigenetic modulators such as DNA methyltransferase inhibitors and/or histone deacetylase inhibitors restores tamoxifen sensitivity in ESR1 negative breast cancer cell lines and opens new treatment horizons in patients who were previously associated with a poor prognosis.In the study presented herein, we tested the ability of ribavirin, which shares some structural similarities with the DNA-methyltransferase inhibitor 5-azacytidine and which is widely known as an anti-viral agent in the treatment of hepatitis C, to restore ESR1 gene re-expression in ESR1 negative breast cancer cell lines.In our study we identified ribavirin to restore ESR1 gene re-expression alone and even more in combination with suberoylanilide hydroxamic acid (SAHA - up to 276 fold induction).Ribavirin and analogs could pave the way to novel translational research projects that aim to restore ESR1 gene re-expression and thus the susceptibility to tamoxifen-based endocrine treatment strategies.

Genomic organization and localization of the NAD-dependent histone deacetylase gene sirtuin 3 (Sirt3) in the mouse.

Sirtuin 3 (SIRT3) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, which belongs to the Silent information regulator 2 (Sir2) family of histone deacetylases (sirtuin HDACs). The yeast Sir2 protein and its mammalian derivatives play a central role in epigenetic gene silencing, DNA repair and recombination, cell-cycle, microtubule organization, and in the regulation of aging. We have isolated and characterized the murine Sirt3 genomic sequence, which spans a region of 18,646 bp and which has one single genomic locus. Determination of the exon-intron splice junctions identified murine SIRT3 to be encoded by 7 exons ranging in size from 101 (exon 4) to 420 bp (exon 7). Characterization of the 5' flanking genomic region, which precedes the murine Sirt3 open reading frame, revealed a number of STATx, GATA and SP1 transcription factor binding sites. A CpG island was not detected. The 1,473-bp murine Sirt3 transcript has an open reading frame of 774 bp and encodes a 257-aa protein (cytoplasmic SIRT3) with a predictive molecular weight of 28.8 kDa and an isoelectric point of 5.82. Recently, a 1,406-bp murine SIRT3 splice variant that encodes a 334-aa mitochondrial precursor protein with a molecular weight of 36.6 kDa and an isoelectric point of 7.19 has been described. Fluorescence in situ hybridization analysis identified a single genomic locus for murine Sirt3 gene on chromosome 7F4 and which is neighbored by the Ric8 and PSMD13 genes. Our study brings light and a number of corrections and additions to previous reports on the genomic organization and the genomic sequence of murine Sirt3, which may be of importance in view of studies on potential genetic polymorphisms in relation to cellular respiration, metabolism, aging-related disease and cancer.