Schizosaccharomyces pombe as model system, we have been able to demonstrate a strong activation of the MAPK Spc1/Sty1 in response to arsenate. This activation is dependent on Wis1 activation and Pyp2 phosphatase inactivation. Using arsenic speciation analysis we have also demonstrated the previously unknown capacity of S. pombe cells to reduce As (V) to As (III). Genetic analysis of several fission yeast mutants point towards the cell cycle phosphatase Cdc25 as a possible candidate to carry out this arsenate reductase activity. We propose that arsenate reduction and intracellular accumulation of arsenite are the key mechanisms of arsenate tolerance in fission yeast.Arsenic toxicity has been studied for a long time due to its effects in humans. Although epidemiological studies have demonstrated multiple effects in human physiology, there are many open questions about the cellular targets and the mechanisms of response to arsenic. Using the fission yeast  Arsenic is a metalloid which is present in the environment both naturally and anthropogenically. As in the case of other metals and metalloids, such as cadmium and chromium, arsenic has been shown to be a health risk at low concentrations. In nature, arsenic is presented in many different oxidation states, being the inorganic ones, arsenite, As (III), and arsenate, As (V), the two main forms. While arsenite is presented mainly in anaerobic and alkaline environments, arsenate is more typical of aerobic and acid environments According to the World Health Organization (WHO), arsenic poisoning is one of the major health problems in several undeveloped countries, although cases have occurred in countries with a higher level of development At intracellular level, both arsenate and arsenite work differently. For instance, arsenate can enter the cell via a phosphate transporter due to its structural similarity to phosphate. For the same reason, arsenate can also alter several biochemical reactions such as cellular respiration. On the other hand, several reports have described the capacity of arsenite to damage DNA since arsenite inhibits base- and nucleotide-excision repair mechanisms Schizosaccharomyces pombe, the stress response is mainly directed by MAPKs and more specifically by the Spc1/Sty1 pathway. Spc1 is analogous to mammalian p38, and is activated when different types of stress such as UV radiation, heat shock and hyperosmolarity are present. In addition, it has been described that p38-like pathways are activated in response to arsenic stress in both S. pombe and Saccharomyces cerevisiaeThroughout the evolution, several mechanisms of response have been developed by organisms against different stressors. For instance, in the fission yeast Escherichia coli Arsb, S. cerevisiae Acr3p, etc). This reducing capacity has been described in unicellular organisms, such as Leishmania major and S. cerevisiae, and pluricellular organisms, such as the fern Pteris vittata and human Regarding arsenate, several reports have shown that MAPK pathway is not the only mechanism of response used by eukaryotic organisms against arsenate. Some organisms can reduce arsenate to arsenite through the activity of arsenate reductases. Arsenite resulting from this reduction is removed from the cell through specific transporters .All strains of For plate survival assays, different concentrations of both arsenate and arsenite (25 µM to 100 µM) were added depending on the experiment. Once the culture reached 0.3 OD, serial dilutions of yeast cultures were spotted in plates. Plates were incubated at 30°C for 48–72 hours.Cells were cultivated up to 0.3–0.5 OD as explained before and arsenate was added at a final concentration of 100 µM. For immunoblotting analysis and mRNA extraction and quantification, cells were harvested by either filtration or centrifugation, respectively, and immediately stored at −80°C.To purify the Spc1:HA6His protein we followed previously described protocol S. pombe cdc25 gene transcription level was determined by a quantitative PCR (qPCR) using that cDNA as template.Cells were harvested by centrifugation at OD = 0.5. Both mRNA extraction and purification were performed as previously described Arsenic speciation studies were performed as described in A Perkin-Elmer 4100 ZL atomic absorption spectrometer with a longitudinal Zeeman background correction, equipped with a transversely heated graphite tube atomizer (THGA) with L’vov platforms was used for arsenic quantification. A Perkin Elmer arsenic electrodeless discharge lamp (EDL) with wavelength 197.3 nm and instrument slit width 0.7 nm was used. A Perkin Elmer EDL System was used to stabilize the lamp current between 349–351 mA. As alternative analytical technique for the determination of As, an ICP-MS HP-7700 Plus  was used. It was equipped with a Babington nebulizer, Fassel torch and double pass Scott-type spray chamber cooled by a Peltier system. Single ion monitoring at m/z 75 was used for data collection.The chromatographic system employed for As speciation consisted of a model PU-2080 Plus Pump,  and PRP-X100 analytical and guard anion-exchange column . The column effluent was directly introduced into the nebulizer of the ICP/MS previously described via a PTFE capillary tube (0.5 mm i.d.). The samples were injected through a six port-valve .3 by dissolving adequate amounts of CH3AsO3Na2 (MMA) and (CH3)2AsO2Na·3H2O (DMA), both 98% purity from Merck . Ten milligrams per liter stock solutions of As (V) and As (III) were prepared fromAs2O5·2H2O (98.5%) from Merck  and As2O3 (99.5%) from J.T. Baker , respectively. All these solutions were kept at 4°C and stored in high density polyethylene (HDPE) bottles until use. Working solutions were prepared daily. The Pd(NO3)2 matrix modifier solution employed for GFAAS analysis was made from a dilution of 10 g/L Pd solution  with water to 2 g/L. HPLC-grade methanol from SDS  and (NH4)H2PO4 from Merck  were the reagents employed as mobile phase of the chromatographic system.High-purity deionized water  was used for sample and standard solutions preparation. Ten milligrams per liter stock solutions, expressed as metal, of MMA and DMA, were prepared in 4% HNOSchizosaccharomyces pombe extract and yeast extract (YES).The samples prepared were kept frozen (−80°C) until analysis. Total arsenic and arsenic species were determined in two types of samples: yeast YES by ZGF AAS by diluting one hundred times and adding five percent of nitric acid to eliminate matrix effects as well as Pd (NO3)2. It was necessary to modify the thermal furnace program respect to recommended conditions by the manufacturer. The furnace program finally employed is showed in 3)2.Total arsenic was determined in Schizosaccharomyces pombe extracts by LC-ICP/MS. The yeast extracts were diluted two hundred times with deionizer water and introduced into a vial Teflon. The ultrasonic probe was then introduced into the solution and sonication was applied during 30 seconds at 30% amplitude. The extracts were centrifuged at 5000 rpm for 10 minutes and the supernatant was passed through a 0.22 µm nylon syringe filter before analysis. The chromatographic conditions were previously optimized . Briefly, a polymeric anion-exchange column, PRP-X100 and mobile phase of 10 mM HPO4−2/H2PO4 at pH 8.5 plus 2% of methanol was added to the 10 mM phosphate mobile. The flow rate was 1 mL/min. Under these conditions appropriate separation of the four targeted species (As (III), MMA, DMA and As (V)) in 9 min can be obtained as shown in a typical chromatogram like Arsenic speciation was carried out in yeast Schizosaccharomyces pombe and cells deficient in this MAPK are sensitive to As (III) Schizosaccharomyces pombe as model organism.We have previously described that trivalent arsenic is able to activate the MAPK Spc1 in First, we monitored the sensitivity to arsenate of different fission yeast strains deficient in one or more genes participating in the activation of the MAPK Spc1 . We compUsing this experimental approach we observed that cells deficient in the MAPKK Wis1 and a double mutant lacking MAPKKKs Wis4 and Win1, were very sensitive to arsenate. However, mutants deficient in Mcs4 or in each one of the MAPKKKs did not show any increased sensitivity to arsenate.These results indicate that the activation of the MAPK Spc1 is essential for the efficient response to arsenate and the activations requires full function of the MAPKK.We had previously described that arsenite activation of Spc1 was mediated through a mechanism that depends on Wis1 activation, but also on a Wis1-activation independent mechanism To further advance in our knowledge of the mechanism of Spc1 activation under arsenate treatment, we also monitored the activation of Spc1 in mutants lacking Wis4 and Win1 activities and each of the phosphatases Pyp1 and Pyp2 .wis4Δ win1-1 genetic background showed a decrease capacity to phosphorylate Spc1 upon arsenate treatment.We reasoned that if the activation of Spc1 was independent of Wis1 phosphorylation, it may be dependent on Pyp1 or Pyp2 inhibition. As seen in This result is consistent with a mechanism where Spc1 regulation is achieved both, through activation of Wis1 and inhibition of Pyp1.Schizosaccharomyces pombe. However, such arsenate reductase activity has not been described in fission yeast yet.The results described above using arsenate as a stress source, resemble those previously obtained with arsenite. One possible explanation for these similar responses could be that arsenate is transformed into arsenite through a biochemical transformation performed by the fission yeast We expected that if such arsenate reductase activity existed in fission yeast, intracellular arsenite should appear in the course of an experimental treatment. We obtained whole cell extracts of fission yeast cells treated with As (V) and determined the intracellular concentrations of As (V) and As (III) at different time points. As seen in in vivo. Our next question was what protein or proteins were carrying out such activity.We have determined that fission yeast presents arsenate reductase activity S. pombe genes similar to known arsenate reductases in other organisms. We found that arsenate reductases and Cdc25 proteins share similarities in their catalytic domain. We compared S. pombe Cdc25 sequence with rice Cdc25 and arsenate reductase Acr2 from Saccharomyces cerevisiae and human arsenate reductase Cdc25.One of our approaches was to look for As observed in cdc2-3w allele, were able to survive in the absence of Cdc25. We therefore monitored the sensitivity of cdc2-3w cdc25Δ strain to arsenate treatment  and As (V) in cellular extracts and growth media obtained from wild type, spc1Δ, cdc2-3w and cdc2-3w cdc25Δ strains, after 3 or 9 hours. This accumulation is specially high in mutants lacking Cdc25, that is consistent with a role of this phosphatase in As (V) removal from fission yeast cytoplasm. After 9 hours, all strains have similar As (V) levels in their cytoplasms. During this period (3–9 hours after treatment), the growth media did show a slight decrease in the total concentration of As (V).In In Together with the accumulation of As (III), these results indicate that Cdc25 might be required for arsenate reductase activity, and that this activity could be affected by the presence of Cdc2 in the cell.S. pombe is able to respond to arsenate have been analyzed. The overall conclusion from these studies is that the response of fission yeast to arsenate and arsenite is different to the response to other types of stress like, for example, high osmolarity. Besides this, we have described for the first time that S. pombe has an arsenate reductase activity. We discuss the possible role of Cdc25 phosphatase as the leading candidate to perform this activity in S. pombe and its possible functional interaction with Cdc2 kinase.In this report, several mechanisms by which S. pombe has a much higher sensitivity to arsenate, reaching growth inhibition at micromolar concentrations  accumulates inside the cells, very little As (III) appears to accumulate in the growth media. This lack of arsenite accumulation could be explained by two different models:The export of As (III) to the cell exterior is not very efficient.There is an spontaneous oxidation of As (III) to As (V) in the growth media.We consider that the second possibility is very unlikely because we have experienced very different responses in sensitivity from fission yeast to arsenate to arsenite. If arsenite would spontaneously oxidized to arsenate, the response to both forms of arsenic would be identical.Therefore, we favour a model where the mechanisms of As (III) removal from the cytoplasm in fission yeast are not based in extracellular elimination, but on vacuolar accumulation, like the mechanism described previously S. pombe. Future research will determine the regulation of this arsenate reductase activity and the possible interplay with other cellular stress response mechanisms.We have described the mechanisms that lead to activation of the MAPK Spc1 by arsenate and the presence of an arsenate reductase activity in


Aspirin plays a crucial physiological and pathophysiological role in cardiovascular diseases and cerebrovascular diseases by irreversibly inhibiting thromboxane A2. However, some patients may be “resistant” to its effect. The resistance has close association with adverse cardiovascular outcomes and increased mortality, so that resolving the problem of aspirin resistance (AR) is widely concerned. By studying the correlation between AR and blood stasis syndrome (BSS), it is demonstrated that BSS may be one of the pathogenesis of AR in traditional Chinese medicine. Chinese herb and formulas definitely possess the advantage of whole body regulation by many ways and many targets. It is a new direction for treatment of AR to combine TCM and modern medicine to study the mechanism and prevention of AR. Despite recent medical advances, cardiovascular diseases (CVDs) remain the primary cause of morbidity and mortality throughout the world . ActivatHowever, some patients may be “resistant” to its effect. AR is widely manifested and associated with increased cardiovascular risk in aspirin-treated patients. This side effect can significantly influence the clinical effect. Measuring response to aspirin is often difficult and there is no accepted definition of AR . On currThere are several proposed mechanisms of aspirin resistance, and true aspirin resistance is unclear. Aspirin noncompliance, insufficient dosing, platelet turnover, and interacting drugs, most notably nonsteroidal inflammatory drugs (NSAIDs), unfortunately are the most important contributors to the treatment failure. Aspirin resistance is a multifactorial phenomenon, so strategies of treatment should be directed to a number of COX-1 dependent and other independent factors, such as increasing patient compliance, increasing dose of levels, combining with other antiplatelet drugs, or applicating other kinds of drugs as alternatives. Also, adequate treatment of confounding clinical conditions such as smoking, hyperlipidemia, hyperglycemia, hypertension, heart failure, infection, and inflammation may further increase efficacy of antiplatelet treatment with aspirin . HoweverWith increasing application of complementary and alternative medicines all over the world, TCM became more popular and have drew more attention –23. It hBy theory of TCM, AR belongs to the category of “collaterals” disease. The pathological character of AR is blood stagnant. Accumulation of blood stasis can be transformed into turbid toxins. Finally, the blood stasis and the turbid toxin congest the “collaterals” and leaded to various diseases. In view of the characteristics of the disease, strategies of PBCRBS and removing toxic materials from meridians should be used to AR. AR will eventually lead to formation of BSS and cause blood stasis in channels and collateral branches by all kinds of reasons. Orient scholars studied the association of AR and BSS and concluded that AR is closely related to BSS –42. BSS Dysfunction of “zang” and “fu” can lead to blood stasis of heart meridian. So that therapeutic of PBCRBS is the first choice of CHD. But, in concrete applications, it is imperative to differentiate TCM syndrome according to the etiology and pathogenesis, the involved zang-fu organs, and clinical manifestation of BSS when using PBCRBS as the main therapy. Also, it is necessary to modulate the liver, the spleen, the lung, and the kidney at the same time and use the corresponding treatment to improve effect of diagnosis and treatment, such as promoting qi circulation, dispelling cold, resolving phlegm, clearing away heat, supplementing qi, supplementing blood, warming yang, nourishing yin, and supplementing kidney. Chinese herbs of PBCRBS have effect on antibacterial, antiviral, inhibiting inflammation, treatmenting infectious diseases, regulating immune system, improving body resistance, and so on . BesidesNowadays, alternative and synergistic treatment of TCM has become an ideal therapeutics. Researches demonstrated that some Chinese herbal compounds  and several traditional medicine monomer or Chinese herbal extract  can effectively inhibit platelet activity. These drugs can be used combining with aspirin to achieve the purpose of enhancing pharmacy efficiency. Also, it can be used as alternative medicine of aspirin for secondary prevention of cardiovascular and cerebrovascular diseases . It is dCurrently, traditional medicine monomer and Chinese formulas used alone or combined with antiplatelet pertensive drugs have been widely used as an alternative and effective method for the treatment of AR with coronary heart diseases in clinical treatment in China. Until now, many clinical studies of formulas used for therapy of AR verified the clinical effect ranging from case reports and case series to controlled observational studies and randomized clinical trials. However, there is no critically appraised evidence such as systematic reviews or meta-analyses on potential benefits and harms of these Chinese formulas for AR to justify their clinical use and their recommendation. This paper aims to assess the current clinical evidence of the frequently used Chinese decoctions or traditional medicine monomer for aspirin resistance in patients with coronary heart diseases.We selected all the clinical trials about Chinese decoctions or traditional medicine monomer for treatment of AR using the search terms of “aspirin resistance,” “coronary heart disease,” “herb,” “Chinese drug,” “compound prescription,” “traditional Chinese medicine,” “decoction,” “Chinese formula,” “controlled clinical trial,” “random,” and “blind” individually or combined in five databases, such as Chinese National Knowledge Infrastructure (CNKI), Chinese Biomedical Literature Database (CBM), PubMed, Embase, and the Cochrane Central Register of Controlled Trials in the Cochrane Library (January 2013). After primary search of five databases, ten RCTs were reviewed –57. All 751 patients with AR were included. Intervention included all the prescriptions of Chinese decoctions or traditional medicine monomer, or combined with antiplatelet-aggregation drugs. The Chinese decoction includes composite salvia dropping pill, Huo xue capsule, Nao xin tong capsule, Tong xinluo capsule, Xuefu zhuyu decoction, and Zhilong huoxue capsule. The traditional medicine extract or monomer can only be reported by five researches , 56, 57,α, platelet counts, and coagulation time. The main finding showed that the combining use of Chinese medicine and antiplatelet-aggregation drugs can decrease the platelet aggregation rate in a different degree more effectively. Of the ten trials, only one research does not show specific data [The treatment duration ranged from 14 days to 1 year. All of the 10 trials used platelet aggregation as the outcome measure. In addition, the outcome measurement also includes the unfrequently used standards, such as uric acid, blood lipid, CRP, hs-CRP, TXB2, COL, 6-K-PGF1fic data .P < 0.00001) and ADP . Also, there was beneficial effect of Chinese formula used alone compared with antiplatelet-aggregation drugs in platelet aggregation rate induced by AA . However, the meta-analysis showed no significant effect of Chinese formula used alone compared with antiplatelet-aggregation drugs in platelet aggregation rate induced by ADP   with BSS, so that etiology and pathogenesis of the disease can be clearly clarified by theory of TCM. Traditionally, the diagnosis of BSS depended on subjective diagnostic methods such as inspection and palpation of the patient . In 1988There are still some problems existing which seriously limited the research and progress on the treatment and should be solved as soon as possible. Researchers of clinical trials in TCM should also pay more attention to experimental design and methodological quality and improve the reporting quality according to the consolidated standards of reporting trials (CONSORT) . Also, i


Variation in polyglutamine repeat number in the androgen receptor (AR CAGn) is negatively correlated with the transcription of androgen-responsive genes and is associated with susceptibility to an extensive list of human disease. Only a small portion of the heritability for many of these diseases is explained by conventional SNP-based genome-wide association studies, and the forces shaping AR CAGn among humans remains largely unexplored. Here, we propose evolutionary models for understanding selection at the AR CAG locus, namely balancing selection, sexual conflict, accumulation-selection, and antagonistic pleiotropy. We evaluate these models by examining AR CAGn-linked susceptibility to eight extensively studied diseases representing the diverse physiological roles of androgens, and consider the costs of these diseases by their frequency and fitness effects. Five diseases could contribute to the distribution of AR CAGn observed among contemporary human populations. With support for disease susceptibilities associated with long and short AR CAGn, balancing selection provides a useful model for studying selection at this locus. Gender-specific differences AR CAGn health effects also support this locus as a candidate for sexual conflict over repeat number. Accompanied by the accumulation of AR CAGn in humans, these models help explain the distribution of repeat number in contemporary human populations. An applied understanding of the evolutionary forces shaping human health and disease susceptibility has profound medical implications, providing clinical insights and suggesting novel, testable hypotheses (Di Rienzo Tandem-repeat polymorphisms can be key components of human phenotypic and disease-related variation (Ashley and Warren Two well-studied human tandem-repeat polymorphisms are situated within the X-linked androgen receptor (AR) gene, which codes for a transcription factor that mediates binding of the androgens testosterone (T) and dihydrotestosterone. Androgens play an integral role in the organizational and ontogenic processes involved in sexual differentiation and male-sexual development during embryogenesis  whose numbers vary both within and between human populations . IndepenFirst, balancing selection contributes to human illness when deleterious alleles are maintained in the population through heterozygote advantage or when disease risk results from extremes of bidirectional variation in gene expression , the online mendelian inheritance in man (OMIM) database , and the genetic association database . All phenotypes associated with polymorphisms in AR CAGn were initially included, but mutations or polymorphisms at other loci in the AR (e.g. AR GGNn) were discarded. Using phenotypes and references obtained from these databases, subsequent searches focused on disease phenotypes and risk associations, and studies investigating the molecular structure and function of the AR were included only for interpreting mechanisms of pathology or their role in selection on AR CAGn frequency in human populations. Additional literature was collected using Web of Science, Google Scholar, and PubMed databases using combinations of the search terms: ‘AR’ or ‘AR’, ‘CAG’, ‘CAG repeat’ or ‘Polyglutamine repeat’, and individual disease names (e.g. ‘Prostate Cancer’).We obtained data on the role of AR CAGn in human health and disease, using three online databases and one comprehensive review  mentioned above and that it be the subject of at least one large-scale (500+ participants) study or meta–analysis examining its relationship to AR CAGn. Diseases that matched these criteria and were the focus of ≥10 independent studies were considered, as was colorectal cancer, for which we deemed the relationship between this disease and AR CAGn to be well supported by two particularly large  case–control studies  shows an unequivocal relationship with AR CAGn. Patients with SBMA invariably have longer repeat number than is observed in the general population, typically between 38 and 62 repeats  are at a 20% increased risk of infertility  Adams , and bet2) with longer AR CAGn  and increase in osteoporosis  risk and progression. Castrated or hypogonadic men (including men suffering SBMA) rarely develop prostate cancer, and chronic exogenous androgen administration in rats can induce the disease  aggression, violence, and criminal activity; (ii) cognitive functioning and general intelligence; and (iii) depression. Other socio-behavioral traits, including social and sexual behavior, have been linked to AR CAGn as well as to other repeat polymorphisms in non-human mammals , which proposes that g increases with repeat length observed along the mammalian lineage is constrained in humans by the negative effects of SBMA and impaired sperm production. This ‘gain in function’ for general intelligence with longer AR CAGn has little empirical support, but given the highly expressed nature of the AR in areas of the brain associated with visual and verbal memory  of repeat length, or at least quantitative proxies for these metrics , the recent construction of a large-scale, annotated database of expressed trinucleotide repeats like the AR CAGn (Luo et al. Still, the challenge of testing evolutionary hypotheses is not restricted to hypotheses of human health and disease (Gluckman et al. These kinds of advances may also provide powerful insights into the role of the AR CAGn in the missing heritability of complex diseases and phenotypes modulated by androgens. Tandem repeats such as the AR CAGn may be more informative than SNPs at the individual level owing to their functional role and greater standing genetic variation in human populations, but have been largely neglected in GWA studies owing to the statistical power and high-throughput assays required to incorporate them (Ku et al. A more comprehensive picture of the heritability of human disease susceptibility must also account for interactions between genes and between genes and the environment (Eichler et al.


Our earlier study indicated that elevated 14-3-3ε expression is significantly associated with higher risk of metastasis and lower survival rates of HCC patients. However, the molecular mechanisms of how 14-3-3ε regulates HCC tumor metastasis are still unclear.In this study, we show that increased 14-3-3ε expression induces HCC cell migration and promotes epithelial-mesenchymal transition (EMT), which is determined by the reduction of E-cadherin expression and induction of N-cadherin and vimentin expression. Knockdown with specific siRNA abolished 14-3-3ε-induced cell migration and EMT. Furthermore, 14-3-3ε selectively induced Zeb-1 and Snail expression, and 14-3-3ε-induced cell migration was abrogated by Zeb-1 or Snail siRNA. In addition, the effect of 14-3-3ε-reduced E-cadherin was specifically restored by Zeb-1 siRNA. Positive 14-3-3ε expression was significantly correlated with negative E-cadherin expression, as determined by immunohistochemistry analysis in HCC tumors. Analysis of 14-3-3ε/E-cadherin expression associated with clinicopathological characteristics revealed that the combination of positive 14-3-3ε and negative E-cadherin expression is significantly correlated with higher incidence of HCC metastasis and poor 5-year overall survival. In contrast, patients with positive 14-3-3ε and positive E-cadherin expression had better prognostic outcomes than did those with negative E-cadherin expression.Our findings show for the first time that E-cadherin is one of the downstream targets of 14-3-3ε in modulating HCC tumor progression. Thus, 14-3-3ε may act as an important regulator in modulating tumor metastasis by promoting EMT as well as cell migration, and it may serve as a novel prognostic biomarker or therapeutic target for HCC. Epithelial-mesenchymal transition (EMT), a developmental process by which epithelial cells reduce cell-cell adhesion and lose apical-basal cell polarity, plays a critical role in the embryogenesis and conversion of early stage tumors into aggressive malignancies via binding intracellular proteins with Ser/Thr phosphorylation-dependent domains, thereby influencing conformation, activity, subcellular localization, and protein complex stability via activation of NFκB signaling via regulation of Zeb-1/E-cadherin expression. Our results reveal that E-cadherin is a downstream modulator for 14-3-3ε during HCC tumor progression.14-3-3 proteins are a family of regulatory molecules with highly conserved homology among all eukaryotic cells 2 at 37°C. For stable transfection, 14-3-3ε cDNA was amplified by PCR and then subcloned into the p3XFlag-CMV vector. Huh-7 cells were transfected with p3XFlag-CMV (Control) or p3XFlag-14-3-3ε (14-3-3ε) by use of Polyjet™ transfection reagent  according to the manufacturer’s instructions. The transfected cells were selected with G418 (500 µg/ml) for 4 weeks. Single colonies of stable clones (at least 3 in each cell line) were maintained in DMEM with 10% FBS and 200 µg/ml of G418.Huh-7 , HepG2 , Hep3B (ATCC-HB-8064), PLC-5 (ATCC-CRL-8024) and SK-Hep1 (ATCC-HTB-52) human hepatocellular carcinoma cells were maintained in DMEM  supplemented with 10% fetal bovine serum , 100 units/ml penicillin, and 100 units/ml streptomycin, in a humidified incubator (Forma) with 5% COHuh-7 and HepG2 cells were transiently transfected with control and 14-3-3ε by use of Polyjet™ transfection reagent . Cells were transfected with control or 0.5 to 1.5 µg of 14-3-3ε vectors per 6-well plate followed by incubation with Polyjet™/DNA complex-containing medium and replaced with complete medium for 24 hours. Transfected cells were incubated for additional 24 hours before performing cell migration assays or protein expression analysis.Gene silencing was performed using 14-3-3ε, Snail , Zeb-1 siRNAs  and Stealth RNA Negative Control  with reported sequences . TransieCells were lysed in ice-cold RIPA buffer  containing cocktail protease inhibitors . Cell lysates were centrifuged at 15,000 rpm for 20 minutes at 4°C, and protein concentrations were determined by a Bio-Rad protein assay kit . Each sample of 20 µg protein was applied to a gradient SDS-PAGE gel and immunoblotted onto PVDF membranes. The membranes were blocked and probed with indicated primary antibodies of Flag and actin , E-cadherin and N-cadherin , 14-3-3ε, Zeb-1, Twist and Slug , Snail , Zeb-2 , and vimentin . The membranes were immersed in PBST containing horseradish peroxidase-conjugated secondary antibody, and protein levels were determined by use of enhanced chemiluminescence reagents.Immunofluorescence staining was performed as described previously 4 for SK-Hep1, 6×104 for Huh-7 and 2×105 for HepG2) were added to the upper wells with 8-µm pores. Cells were allowed to migrate toward the bottom wells containing 100 µg/ml fibronectin , epithelial growth factor  and 10% BSA-DMEM for 20 hours. Cells remaining on the upper side were removed, and migrated cells on the bottom side were fixed and stained with 0.1% crystal violet containing 20% ethanol and 1% formadehyde for 20 minutes. Cell migration was quantified by counting the total number of migrated cells.Bio-coat cell migration Boyden chambers were used for cell migration assay . Briefly, cells were trypsinized and suspended in 0.1% BSA-DMEM and cells . Quantitative real-time PCR involved use of SYBR Green  with specific oligonucleotide primers  developed tissue-proved metastasis in 3 to 87 months after the resection of primary HCC. Slides from paraffin-embedded surgical specimens of primary tumors with surrounding non-cancerous liver parenchyma were subjected to immunohistochemical (IHC) staining. The pathological features, IHC staining results, clinical parameters, including Barcelona-Clinic Liver Cancer (BCLC) staging For immunohistochemistry analysis, an automatic immunostaining device and ultraView detection kit  were used to detect 14-3-3ε expression in paraffin-embedded tissues by use of a primary antibody against 14-3-3ε  and E-cadherin . A negative control was prepared by the same staining procedure without primary antibodies. The intensity of 14-3-3ε and E-cadherin protein staining was semiquantitatively scored by a Quick-score (Q-score) method based on intensity and heterogeneity t-test was used to analyze differences between 2 groups. Kaplan-Meier curves were plotted and the log rank test was used to analyze time-related variables of probabilities for metastasis and overall survival. A P value <0.05 was considered statistically significant.The Student’s To explore the potential role of 14-3-3ε in HCC tumor metastasis, we examined the expression of 14-3-3ε in distinct HCC cell lines. 14-3-3ε was detected in all tested HCC cells . The welTo investigate whether 14-3-3ε expression regulates EMT of HCC cells, we determined the expression of EMT markers, E-cadherin, N-cadherin and vimentin, by Western blotting analysis. We found that 14-3-3ε overexpression significantly reduced E-cadherin expression, but it induced N-cadherin and vimentin expression . The expTo understand the molecular regulation of how 14-3-3ε induces EMT and reduces E-cadherin expression in HCC, we examined the expression levels of distinct E-box transcriptional suppressors. We found that 14-3-3ε overexpression selectively induced Zeb-1 and Snail expression but had no significant effect on Zeb-2, Twist or Slug . The indTo further explore the role of Zeb-1 and Snail on 14-3-3ε-induced cell migration or EMT, we knockdown Zeb-1 and Snail with siRNAs and examined E-cadherin expression by Western blotting analysis. Interestingly, 14-3-3ε-reduced E-cadherin expression was specifically restored by Zeb-1 siRNA, but not by Snail siRNA . This spp = 0.043)  . In addi= 0.043) .p = 0.011), histology grade (p = 0.001), BCLC staging (p = 0.030), tumor size (p = 0.003), and subsequent extrahepatic metastasis (p = 0.004) (p = 0.013) and better overall survival rate (p = 0.047) than do those with negative E-cadherin expression in 14-3-3ε positive HCC tumors. These results provide clinical evidence to support the hypothesis that E-cadherin is one of the crucial downstream regulators of 14-3-3ε that modulate HCC tumor progression.We have previously shown that 14-3-3ε overexpression in HCC primary tumors was significantly associated with subsequent extrahepatic metastasis and reduced 5-year overall survival = 0.004) . Patientvia upregulation of Zeb-1. We found that the expression of Zeb-1 was increased (14 of 113) in HCC primary tissues , SMAD2, and PLA2G2A were regulated in 14-3-3ε overexpression cells but had a reversed expression pattern in Zeb-1 knockdown cells (Data not shown). These results provide additional evidence to support our findings.Expression of transcriptional repressors for E-cadherin, including Zeb-1, Snail, SIP1 and Twist, is associated with cell invasion, EMT, metastasis and poor patient survival of HCC via activation of NFκB to enhance HCC cell migration In addition to Zeb-1, our results indicated that 14-3-3ε induces Snail expression and promotes HCC cell migration . HoweverFigure S1Representative immunohistochemical analysis of 14-3-3ε and Zeb-1 in HCC tissues. 14-3-3ε and Zeb-1 are positive expressed in HCC tumors.(TIF)Click here for additional data file.Figure S2Smad-2 does not regulate 14-3-3ε/E-cadherin expression. Smad-2 siRNA has no significant effect on the restoration of 14-3-3ε-reduced E-cadherin expression.(TIF)Click here for additional data file.Figure S3TGF-β and PI3K/Akt do not regulate 14-3-3ε/E-cadherin expression. Treatment with SB431542 (TGF-βR Inhibitor) or LY294002 (PI3K Inhibitor) has no significant effect on the restoration of 14-3-3ε-reduced E-cadherin expression.(TIF)Click here for additional data file.Figure S414-3-3ε does not direct interact with Zeb-1. No significant interaction between 14-3-3ε and Zeb-1 was observed by using co-immunoprecipitation. Cell lysates were collected and subjected to Protein A magnet beads (Millipore) immunoprecipitation followed by Western blot analysis.(TIF)Click here for additional data file.Table S1Oligonucleotide sequences of small interfering RNAs. siRNA sequences for YWHAE (14-3-3ε), SNAI1 (Snail) and ZEB1 (Zeb-1).(TIF)Click here for additional data file.Table S2Oligonucleotide sequences for Q-PCR procedures. Primer sequences for CDH1 (E-cadherin), SNAI1 (Snail), ZEB1 (Zeb-1), SMAD2 (Smad-2) and PLA2G2A.(TIF)Click here for additional data file.Table S3Correlation of E-cadherin expression with 14-3-3ε and clinicopathological characteristics in primary HCC patients. One-way ANOVA was used to analyze correlation between clinicopathological parameters and 14-3-3ε with E-cadherin expression.(TIF)Click here for additional data file.


PSA gene. Data from ectopic overexpression and “knock-down” approaches demonstrated that AR transcriptional activity was enhanced by FUS. Depletion of FUS reduced androgen-dependent proliferation of LNCaP cells. Thus, FUS is a novel co-activator of AR in prostate cancer cells.Androgen receptor (AR) is a member of the nuclear receptor family of transcription factors. Upon binding to androgens, AR becomes transcriptionally active to regulate the expression of target genes that harbor androgen response elements (AREs) in their promoters and/or enhancers. AR is essential for the growth and survival of prostate cancer cells and is therefore a target for current and next-generation therapeutic modalities against prostate cancer. Pathophysiologically relevant protein-protein interaction networks involving AR are, however, poorly understood. In this study, we identified the protein FUsed/Translocated in LipoSarcoma (FUS/TLS) as an AR-interacting protein by co-immunoprecipitation of endogenous proteins in LNCaP human prostate cancer cells. The hormonal response of FUS expression in LNCaP cells was shown to resemble that of other AR co-activators. FUS displayed a strong intrinsic transactivation capacity in prostate cancer cells when tethered to basal promoters using the GAL4 system. Chromatin immunoprecipitation experiments showed that FUS was recruited to ARE III of the enhancer region of the  Androgen receptor (AR) is required for the survival and growth of prostate cancer cells. Accordingly, advanced prostate cancer can be treated with androgen ablation therapies. Unfortunately, these therapies eventually fail and the disease becomes lethal, castration-resistant prostate cancer (CRPC). The most widely supported mechanism underlying CRPC involves AR. The amino-terminus domain (NTD) of AR is required for both ligand-dependent and ligand-independent activity of the receptor  at their NTD and RNA recognition motif (RRM) and repeats of the tripeptide RGG at their carboxyl terminus In vivo association of FUS with AR was confirmed using extracts of LNCaP xenografts that were grown in mice before or after castration -luciferase reporter gene construct. This reporter contains the PSA promoter and enhancer regions harboring several AREs that confer induction by androgens PSA mRNA and mRNA from five other androgen-inducible genes were significantly reduced upon depletion of FUS . Consistent with reduced levels of mRNA, levels of PSA protein were significantly reduced in LNCaP cells upon depletion of FUS , however, had negligible effects on expression of FUS expression  is modulated by androgens and/or AR n of FUS .To determine if the expression of FUS is altered in cancer relative to benign prostate tissue, Oncomine database analysis was performed using seven different clinical cohorts. Expression of FUS was elevated in prostate carcinoma compared to benign tissue using data from three studies . Howeverin vitro and in vivo; and (7) levels of FUS were elevated in clinical samples of prostate cancer.AR is essential for all phases of prostate cancer progression including the terminal CRPC stage. Underlying this role of AR is a dynamic interaction with a various co-activators and co-repressors. The scope of pathophysiologically relevant protein-protein interaction involving AR in prostate cancer cells is, however, not fully known. Here, we show for the first time that FUS interacted with AR endogenously in prostate cancer cells and further revealed the following: (1) FUS had intrinsic transactivation capacity in prostate cancer cells; (2) FUS enhanced AR-mediated transcription; (3) depletion of FUS reduced levels of expression of androgen-induced genes; (4) FUS was recruited to AREs in response to androgen; (5) reduction of levels of FUS limited androgen-induced proliferation; (6) levels of FUS were altered by androgen status The DNA-binding domains (DBD) steroid receptors display high sequence homology , and can interact with many of the same coactivators. Using recombinant proteins, FUS has been shown to interact with the DBDs of retinoid X, estrogen, thyroid, and glucocorticoid receptors, but AR was not examined Here, we demonstrate that FUS harbors strong transactivation domains that were functional in prostate cancer cells. The C-terminal region containing the RRM and RGG motifs of EWS can suppress the transactivation capacity of the NTD in rabbit kidney-derived RK-13 cells in vitro, and in vivo castrate levels of androgen were associated with increased levels of FUS. This expression profile resembles that of previously characterized AR co-activators that are modestly repressed by androgens Expression of various co-regulators of AR is responsive to androgens perhaps as part of feed-back mechanisms AR gene disruptions AR transcriptional activity is required for the maintenance and growth of the prostate which forms the rationale for androgen ablation therapies for prostate cancer. Consistent with FUS being a coactivator of AR and its depletion reducing AR transcriptional activity and androgen-dependent growth as shown here, among the prominent phenotypes in FUS−/− mice is male sterility, and smaller male reproductive organs with apparent involution of some of these structures 1) FUS interacts with AR; and 2) FUS enhances the transcriptional activity of AR.Overall, while further studies are needed to resolve the role of FUS in prostate cancer disease progression, this study revealed that: probasin gene fused to the basal thymidine kinase (TK) promoter FUS expression plasmids LNCaP cells, obtained from Dr. Leland W.K. Chung , were grown in RMPI containing 5% Fetal Bovine Serum (FBS) and were used at passage 39–50. HEK293 cells were maintained in DMEM containing 10% FBS. Unless stated otherwise, cells were incubated in serum-free, phenol-red free media for 24–48 hrs prior to treatment with indicated concentrations of R1881. Transfection of LNCaP cells was performed using lipofectamine 2000 (Invitrogen) for experiments involving siRNAs or lipofectamine (Invitrogen) for all others as per manufacturer's instructions at 0.2–0.4% and 0.5% (v/v), respectively. FUS  and control siRNAs (Invitrogen) were used at 10–50 nM concentrations. HEK293 cells were transfected using lipofectamine 2000 at 0.4% v/v. Exact amounts of plasmids per transfection are indicated in Figure legends.GAPDH levels according to the Pfaffl method. FUS primers Total RNA was prepared using the RNeasy mini kit (Invitrogen). Total RNA (0.5 µg) was reverse-transcribed via oligo-dT priming using the SuperScript III kit (Invitrogen). PCR was performed using gene-specific primers in the presence of SYBR Green Supermix (Invitrogen) using the ABI 7900 real-time PCR machine . The thermocycling protocol was as follows: at 95°C for 2 min, followed by 40 cycles of 95°C for 15 sec and 55°C for 30 sec. Ct (threshold cycle number) values were converted to mean expression values relative to Cells were washed with PBS and fixed in 1% paraformaldehyde in PBS for 30 min. Cells were permeabilized in 0.1% Trition X-100 in PBS twice for 5 min. After blocking for 30 minutes in 2% BSA in PBS containing 0.1% Trition X-100, samples were incubated with the indicated antibodies (1∶50) in blocking buffer for 1 hr. Cells were washed with blocking buffer 4 times and incubated with appropriate secondary FITC or rhodamine-conjugated IgG (1∶100) in blocking buffer for 45 min. After 4 washes with PBS containing 0.1% Trition X-100, DAPI-containing mounting solution was added. Slides were visualized using using Fluoview confocal microscope .6) were plated in 15 cm dishes with RPMI containing 5% FBS. After 24 hrs, media was changed into serum- and phenol red-free RPMI and the cells were incubated for additional 24 hrs. Cells were subsequently treated with R1881 (10 nM) or its vehicle (ethanol) for 6 hrs. Cells were rinsed with PBS before harvesting and then lysed with 1 ml of lysis buffer  in the presence of a cocktail of protease inhibitors . Lysates were passed through 27-G needles five times and were subsequently pre-cleared with protein A/G agarose beads (5% v/v) and a mixture of mouse and rabbit IgG (2 µg/ml each) for 1 hr. The resulting supernatant was incubated with the indicated specific antibodies including AR 441, AR C19, and FUS  at a concentration of 1–2 µg/ml for 16–24 hrs. Protein A/G agarose beads (5% v/v) were subsequently added and the mixture incubated for an additional 3 hrs. The beads were washed with 1 ml of the lysis buffer five times and then resuspended in 40–80 µl of standard SDS sample buffer.LNCaP cells  according to standard protocols using Tris/glycine-based transfer buffer in Bio-Rad's submarine system . Blots were probed with following antibodies: AR PG21 (1∶1000) ; AR 441 (1∶500), or FUS 4H1 (1∶1000) (Santa Cruz Biotechnology Inc).5) in 10 cm plates were transfected with siRNAs using lipofectamine 2000 (Invitrogen). After 24 hrs, the media was changed into RPMI containing 10% charcoal-stripped FBS and cells were incubated for additional 24 hrs before treatment with 0.1 nM R1881 or equivalent amount of ethanol. Two days pos-treatment, cells were allowed to incorporate BrdU  at 10 uM for 3–4 hrs. Subsequently, cells were harvested, washed with PBS and fixed with ethanol at −20°C for a minimum of 2 hrs. Cells were washed and their DNA was denatured with 2 N HCL, 0.5% Trition X-100 for 20 min. Following 4 washes, cells were blocked with 4% FBS/0.1% Trition X-100 in PBS and subsequently stained with 1∶40 ant-BrdU antibody. After two washes, DAPI was added at 2 ug/ml and samples were subjected to flow cytometry.LNCaP cells , and tissue processing thereafter was conducted as previously described Figure S1Identification of FUS as AR -interacting protein by co-immunopreciptation followed by mass spectrometry (MS). MS/MS spectrum of m/z 2251.86  of FUS was unambiguously assigned the identified sequence APKPDGPGGGPGGSHMGGNYGDDR. Spectra for two other peptides are shown in (TIF)Click here for additional data file.Figure S2Identification of FUS as AR -interacting protein by co-immunopreciptation followed by mass spectrometry (MS). MS/MS spectrum of m/z 1407.64  of FUS was unambiguously assigned the identified sequence LKGEATVSFDDPPSAK. A third peptide spectrum is shown in (TIF)Click here for additional data file.Figure S3Comparative analysis of FUS expression in various cancer cells. Cell lines representing various cancer types were grown in their respective cell lines. Quantitative RT-PCR was performed to quantify FUS mRNA relative to GAPDH. LNCaP was used as reference for relative normalized expression. Prostate cancer cell lines that are AR positive are shown in red and in blue are AR negative prostate cancer cell lines. In black are non-prostatic cancer cell lines. Columns = mean ± standard deviation. *P<0.05, n = 3.(TIF)Click here for additional data file.Figure S4Comparative analysis of FUS expression in clinical samples from various cancer types. Data from a previous study is mined using Oncomine.(TIF)Click here for additional data file.


In summary, results of the present study indicate that the status of AR expression levels in UUTUCs may be a significant factor in affecting the efficacy of chemotherapy and classic chemotherapeutic drugs and AR targeted therapy may provide a novel potential therapeutic approach to improve treatment of UUTUCs.Upper urinary tract urothelial carcinomas (UUTUCs) represent relatively uncommon yet devastating tumors that affect more males than females. However, the correlation between gender difference and disease progression remains unclear. Androgen and the androgen receptor (AR) were previously hypothesized to account for the gender difference in the incidence of urothelial carcinomas; however, the role of AR in the development and progression of UUTUCs is not well understood. In addition, although UUTUCs are responsive to chemotherapy, various responses are presented among patients. Therefore, the aim of the present study was to determine the role of AR in the response of UUTUC cells to chemotherapeutic drugs. In this study, AR overexpression in UUTUC cells (BFTC 909) was identified to reduce the cytotoxic effect of chemotherapeutic drugs, including doxorubicin, cisplatin and mitomycin C and protected cells from drug-induced death. The expression of ABCG2, an ATP-binding cassette half-transporter associated with multidrug resistance, was increased in AR-overexpressing BFTC cells. In addition, use of the AR degradation enhancer, ASC-J9 Upper urinary tract urothelial carcinomas (UUTUCs) are relatively uncommon tumors, accounting for ∼5% of all urothelial and 5–10% of all renal tumors. However, UUTUCs are associated with severe morbidity and mortality . These cIn a previous study, the 5-year disease-specific survival rates of the patients by primary tumor stage were identified as 100% for Ta/cis, 91.7% for T1, 72.6% for T2 and 40.5% for T3. Patients with primary stage T4 tumors had a median survival of 6 months ,12. SincUUTUC affects more males than females with a male-to-female ratio of 3:2 for tumors in the renal pelvis and 2:1 for those in a ureteral location . Diseasein vitro and xenograft tumor growth in vivo(The role of androgen receptor (AR) in urothelial carcinoma of the ureter and renal pelvis remains unclear; however, AR has been demonstrated to affect urothelial carcinoma of the bladder . AR was h in vivo. Furtherh in vivo. These oh in vivo. However2, was kindly provided by Dr Tzeng  and cultured in Dulbecco’s modified Eagle’s medium, containing 10% heat-inactivated fetal bovine serum (FBS) at 37°C in an atmosphere of 5% CO2.μg/ml puromycin and AR-overexpressing clones  were named BFTC 909 pBabeAR1 and BFTC 909 pBabeAR2.To establish BFTC 909 cell lines overexpressing AR, cells were stably transfected with a pBabe-hAR plasmid using Lipofectamine  following the manufacturer’s instructions. Cells were selected using 1 To exogenously express AR, a recombinant lentiviral vector containing wild-type AR (pWPI hAR) and a control lentiviral vector expressing the enhanced green fluorescent protein (pWPI) were used to overexpress AR. Lentiviral PWPI-AR/PWPI-control with pMD2.G packaging and psPAX2 envelope plasmids  were co-transfected into 293T cells. Following 48 h transfection, the target cells were cultured in the presence of viral supernatant containing 8 mg/ml polybrene  for 6 h. Flow cytometry was used to analyze cells overexpressing AR and dead cells were evaluated by propidium iodine (PI) under chemotherapeutic drug treatment.® -1,4,6-heptatrien-3-one) was donated by AndroScience Corp. .Cisplatin (P4394) was purchased from Sigma-Aldrich . Doxorubicin hydrochloride and mitomycin C were obtained from the Hospital Pharmacy at China Medical University Hospital, Taiwan, China. ASC-J94 cells/well. After 24 h, complete medium was removed and changed to DMEM containing 10% FBS and cisplatin, doxorubicin or mitomycin C at designated concentrations. After 48 h, fresh DMEM containing 50 μl XTT solution was added directly to the medium and cells were incubated for an additional 3 h at 37°C. A test wavelength between 450–500 nm and a reference wavelength of 650 nm were used in the assay.Viability of BFTC 909 cells was evaluated in 96-well plates 48 h after treatment by measuring cellular viability using the Cell Viability kit (XTT) . The cytotoxic effects of chemotherapeutic drugs were determined by the colorimetric XTT assay based on the activities of mitochondrial enzymes in viable cells. Cells were seeded into 96-well culture plates at a seeding density of 1x10μg/lane) were electrophoretically separated and transferred onto nitrocellulose. Membranes were incubated with antibodies against AR or ABCG2 , followed by horseradish peroxidase-conjugated secondary antibody. Protein-antibody complexes were detected by an enhanced chemiluminescence system  using the Bio-Rad imaging system .For western blot analysis, protein extracts of each sample  according to the manufacturer’s instructions. Total RNA was reverse transcribed into cDNA using BluePrint RT Reagent Kit . The primer sequences were as follows: human AR, forward 5′-TGT CCA TCT TGT CGT CTT C-3′ and reverse 5′-CTC TCC TTC CTC CTG TAG-3′; human β-actin, forward 5′-TCA CCC ACA CTG TGC CCA TCT ACG A-3′ and reverse 5′-CAG CGG AAC CGC TCAT TGC CAA TGG-3′; and human ABCG2, forward 5′-GGG TTC TCT TCT TCC TGA CGA CC-3′ and reverse 5′-TGG TTG TGA GAT TGA CCA ACA GAC C-3′. qRT-PCR was performed using the Bio-Rad CFX96 real-time thermal cycler and SYBR Premix. Relative mRNA expression levels were normalized against β-actin  and determined by the 2μl PI (0.5 mg/ml) for 10 min at room temperature. Cell death was analyzed by flow cytometry .PI exclusion was used to measure cell death. Cells were treated with various drugs for 48 h at 37°C. After 48 h, cells were trypsinized and resuspended in 1 ml PBS with 20 Statistical analysis was performed using Microsoft Excel using a two-sided Student’s t-test. Data are presented as mean ± SD. P<0.05 was considered to indicate a statistically significant difference.To determine the role of AR in the progression of UUTUCs, AR was overexpressed by stably transfecting AR cDNA into the UUTUC cell line, BFTC 909 . Two celTo explore the effect of AR on cancer cells, the effect of AR on the cytotoxic effect of chemotherapeutic agents was determined. Adjuvant systemic chemotherapy is known to provide therapeutic benefit in patients and prevent recurrent bladder tumors with invasive UUTUCs ,24. CispTo further determine the effect of AR on UUTUC cells treated with chemotherapeutic drugs and reduce the potential artificial effects via overexpression AR using plasmids, BFTC 909 cells were transfected with a lentiviral system carrying pWPI hAR or control pWPI parental vectors expressing green fluorescent protein (GFP) and treated with doxorubicin . With siThe membrane transporter protein ABCG2 is linked to multi-drug chemoresistance in cancer cells due to its ability to reduce the intracellular concentrations of anticancer drugs using the energy of ATP hydrolysis to transport drugs across the cell membrane . To deteObservations of the present study indicate that it may be possible to alter the response of UUTUC cells to the cytotoxic effect of chemotherapeutic drugs via alteration of AR expression in the cells to enhance their destruction. ASC-J9, the first AR degradation enhancer to be identified that degraded AR in selective cells with few side-effects ,27–30 waPrevious studies have hypothesized that gender is a factor in the incidence and progression of UUTUCs due to the observation that males have a substantially higher risk of developing UUTUCs. In addition, it has been identified that females tend to have more aggressive tumors ,3,11. Alet al demonstrated the role of androgen and AR in chemically induced mouse bladder cancer and identified that androgen and AR are involved in the carcinogenesis of bladder carcinoma, indicating that AR may be an androgen-independent carcinogenesis factor for bladder cancer development (in vitro and in vivo(Although several studies examined the AR expression pattern in human bladder cancer, the prognostic significance of AR expression is inconsistent with studies demonstrating that loss of AR expression is associated with invasive bladder cancer ,18 or haelopment . It is ld in vivo.Chemotherapy is used to increase lifespan and prevent disease recurrence following surgical resection of localized cancer. Chemotherapy is also utilized as part of the multimodal treatment of locally advanced cancer, allowing for more limited surgery with organ sparing and even cure. Therefore, more effective and less toxic chemotherapy regimens are likely to significantly benefit cancer patients. Cisplatin and doxorubicin have been previously used in adjuvant chemotherapy regimens for patients with upper tract transitional cell carcinoma and a positive outcome was reported ,32. MitoFurthermore, the role of AR in promoting cancer is well characterized in prostate cancer with multiple mechanisms involved, including cell cycle regulation, apoptosis and kinase signals . HoweverTo explore the clinical application of AR signaling on the treatment of UUTUC, ASC-J9, an AR degradation enhancer, which selectively targets AR without affecting libido, fertility and sexual behavior, was used. Our previous study demonstrated that ASC-J9 treatment led to degradation of AR in a number of cell lines and the effect was AR-specific. ASC-J9 was revealed to have therapeutic effects on spinal bulbar muscular atrophy mice via degradation of AR without affecting serum testosterone levels . ASC-J9 In the present study, AR was demonstrated to play a role in the suppression of the cytotoxic effect of doxorubicin, cisplatin and mitomycin C treatment to UUTUC cells, indicating that the AR expression status of UUTUCs affects chemotherapeutic efficacy. In addition, AR was identified as a novel therapeutic target in UUTUCs to increase the efficacy of chemotherapeutic agents. Therapies targeting the androgen-receptor signaling axis have been used for the treatment of prostate cancer with various strategies ,43 and m


To test, whether 10 genes, diagnostic of renal allograft rejection in blood, are able to diagnose and predict cardiac allograft rejection, we analyzed 250 blood samples from heart transplant recipients with and without acute rejection (AR) and with cytomegalovirus (CMV) infection by QPCR. A QPCR-based logistic regression model was built on 5 of these 10 genes (AR threshold composite score>37% = AR) and tested for AR prediction in an independent set of 109 samples, where it correctly diagnosed AR with 89% accuracy, with no misclassifications for AR ISHLT grade 1b. CMV infection did not confound the AR score. The genes correctly diagnosed AR in a blood sample within 6 months prior to biopsy diagnosis with 80% sensitivity and untreated grade 1b AR episodes had persistently elevated scores until 6 months after biopsy diagnosis. The gene score was also correlated with presence or absence of cardiac allograft vasculopathy (CAV) irrespective of rejection grade. In conclusion, there is a common transcriptional axis of immunological trafficking in peripheral blood in both renal and cardiac organ transplant rejection, across a diverse recipient age range. A common gene signature, initially identified in the setting of renal transplant rejection, can be utilized serially after cardiac transplantation, to diagnose and predict biopsy confirmed acute heart transplant rejection. Despite improvements in immunosuppressive therapy over the years, approximately 30–40% of heart transplant recipients require treatment for acute rejection (AR) in the first year after transplantation Recent studies indicate that there likely is a common immunological mechanism for AR across different solid organ transplants The present study design is summarized in QPCR discovery set. A multinomial logistic regression model using 5 genes, built in the training-set and validated in the test-set was secondly applied to an independent QPCR validation set of 86 blood samples with matched EMB. The model was tested (1) for its ability to segregate samples with AR from those without any evidence of rejection; it was tested (2) for its ability to discriminate AR from acute CMV infection in 12 blood samples from patients with documented active CMV infection; and the model was tested (3) for its ability to predict the development of CAV at 2- and 4-years post transplantation in patients with biopsy proven AR at 1 year, as AR is an important risk factor for the development of CAV. Finally, serial blood samples were available from 23 patients that were drawn within 6 months prior to or after an episode of biopsy-confirmed AR. The 5 gene model was tested in this QPCR prediction-set to ascertain the “rejection score”, to determine whether the gene expression score rose prior to episodes of biopsy-proven AR, and whether the score declined after treatment of the rejection event.To investigate, whether the same 10-gene signature may also be modulated in cardiac AR, we investigated 141 peripheral blood samples with matched EMB, from adult heart transplant recipients by QPCR . FirstlyAll patients involved in this study provided informed consent to the study protocol approved by the institutional (Stanford University) review board for studies in human subjects.This biomarker study utilized a cohort of 45 consecutive patients undergoing first heart transplantation between January 2002 and May 2005 at Stanford University. This cohort was a selected subset of samples from a clinical trial that was funded by the National Institutes of Health (Program Project Grant (PPG) 5P01AI050153-02) and had been assembled prospectively to study the relationship between CMV infection and the development of CAV. Exclusion criteria for this trial included age <10 years, renal dysfunction requiring prolonged dialysis, and inability or unwillingness to provide signed informed consent. Study patients in this trial had been monitored for acute cellular rejection by surveillance EMB performed at the following scheduled intervals after transplant: weekly during the 1st month, biweekly until the 3rd month, monthly until the 6th month, and then at months 9 and 12. Biopsies were graded according to the 1990 International Society for Heart and Lung Transplantation (ISHLT) classification system as 0, 1A, 1B, 2, 3A, and 3B  [18]. ThIn the study, yearly coronary angiograms were performed with intravascular ultrasound (IVUS), enabling highly accurate measurements of vessel wall thickness, for assessment of CAV which is characterized by diffuse intimal thickening of the graft coronary arteries Peripheral whole blood samples were collected and stored at the following time-points post-transplant in the parent PPG: day 14; months 1, 2, 3, 4, 5, 6, 9, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, and 60. For demographic characteristics of patients included in the study refer to Post-transplant immunosuppression consisted of Daclizumab (1 mg/kg IV) administered at the time of transplant surgery and on alternate weeks for a total of five doses, Cyclosporine (3–5 mg/kg/day); Prednisone initiated at 1 mg/kg/day and tapered to <0.1 mg/kg/day by the 6th post-operative month; and either Mycophenolate mofetil 1000–3000 mg daily, or Sirolimus 1–4 mg daily. Changes to this standard immunosuppressive regimen were made on an individual basis. All patients in whom either donor or recipient was CMV antibody positive received standard CMV prophylaxis consisting of 4 weeks of intravenous Ganciclovir. Those recipients who were CMV antibody negative and received a heart from a CMV antibody positive donor received an additional 3 months course of CMV hyperimmune serum and up to 80 days of Valganciclovir.2 First Strand cDNA Synthesis Kit (Bioscience), followed by quantitative real-time polymerase chain reaction (QPCR) in 384-well plates using the QPCR Master Mix (RT2 SYBR Green/ROX) (Bioscience). 5 ng cDNA were added to each 10 µl QPCR reaction in duplicated wells. QPCR reactions were run on the ABI PRISM 7900HT Sequence Detection System . The relative amount of RNA expression was calculated using comparative CT method Peripheral blood (2.5 mL) was collected into PAXgene™ Blood RNA tube  containing lysis buffer and RNA stabilizing solution. Total RNA was extracted with the PAXgene™ Blood RNA System  following the manufacturer's instructions and as previously published Selection of the 10 genes for gene expression analysis in this study was done through a multi-platform microarray discovery followed by QPCR validation in kidney transplantation Mean ± standard deviations of were calculated for patient demographic variables, and mean ± standard errors of means were determined for QPCR results. Student T-tests, Chi-square tests, Hypergeometric tests QPCR-generated gene expression data for a set of 10 genes , originally identified and validated in 458 peripheral blood samples from pediatric recipients of a renal transplant The fixed 5 gene-model was subsequently tested in 86 independent samples with varying AR grades, STA and CMV diagnoses, and identified AR samples of all grades <3 with 89% accuracy  and at 4 years  post-transplantation. Furthermore, predicted probabilities of AR at 1 year were significantly higher in patients with higher grades of CAV (CAV score≥3) vs. mild grades of CAV (CAV score≤2) at 4 years post-transplantation , which indicated that patients with higher predicted AR probability, independent of AR histology grade, may be at greater risk to develop more severe CAV at subsequent follow-up.No significant demographic or clinical variables  were found to be confounders for the ability of the 5-gene model to diagnose AR (maximum |r|<0.4 or p>0.05), and specifically time-post transplant for sampling did not confound the score, which has been an issue in other biomarker studies of this nature after treatment of acute rejection varied between (0%–100%), with an average prediction score of 87%  and sensitivity for ruling-in the presence of AR. The clinical utility of a blood gene profiling approach for ruling out AR was demonstrated in a randomized study on 600 heart transplant recipients, where there was non-inferiority of an Allomap-based rejection monitoring strategy, compared to EMB, with respect to a composite endpoint of AR, graft failure and death, and a reduction in the number of EMBs performed in this study by almost 70%, consistent with the high NPV associated with the Allomap test ruling-in the presence of AR in the same study suggested that complementary approaches for the diagnosis and prediction of AR, such as the use of the present 5-gene panel by this study, are needed.The Cardiac Allograft Rejection Gene expression Observational (CARGO) study Although management of heart transplant recipients often varies between centers, most transplant programs only consider rejection of Grade 3A or 3B (showing myocyte damage) as clinically relevant, and therefore warranting treatment. Currently, AR of grades of 1A, 1B and 2 are frequently dismissed, without any additional treatment delivery, perhaps because these lower histological grades of rejection are observed so commonly in the protocol biopsies performed. Interestingly, the inflammatory infiltrate that is common to all histological grades (1–4) of AR and is singularly absent in the non-rejection biopsies (Grade 0), suggests that the presence of an infiltrate is a very common finding, and in the absence of myocyte damage its clinical relevance in heart transplantation remains unclear. Nevertheless, the presence of an inflammatory infiltrate of predominantly mononuclear cells is the hallmark of AR in other solid organ transplants such as kidney et alpost hoc analysis of the CARGO data, specifically examining gene expression scores for blood samples accompanying EMB of varying grades. They demonstrated that the mean gene expression scores for Grades 1B and ≥3A were indistinguishable, once again suggesting a potential overlap along a molecular spectrum of rejection severity. A recent study by Holweg et al. The 5-gene model developed and tested in this study can diagnose acute cardiac rejection of Grades 1A–3B (no Grade 4 samples were available for this study), with the highest confidence for diagnosing Grade 1B rejection. Molecular subtyping has demonstrated evidence of myocyte apoptosis in Grade 1B biopsies that is a feature of myocyte damage typical of Grade 3A biopsies, but not of less severe (Grade 1A) rejection predict the onset of AR, months before EMB based histological diagnosis. Importantly, the AR probability score defined by our 5-gene model decreased again after augmented immunosuppressive therapy in patients with rejection grades 3A/B, and remained elevated in untreated cases of AR of grades≤2. Further studies are required to evaluate the prediction probability of this gene-set as a means to titrate immunosuppression in heart transplantation, without the need for frequent protocol biopsies.The 5-gene model developed in this study could also Our previous work in kidney transplantation Although these results are intriguing, they mandate further validation in larger, prospective cohorts, as the sample set in this study was small and the analysis was retrospective in nature. The gene panel demonstrated the utility of distinguishing the presence of cellular rejection  from immunologic quiescence; however, further refinement of our algorithm is planned to distinguish clinically-relevant grades of rejection from those that do not require treatment. Additionally, this study focused on the ability of our model to diagnose acute cellular rejection, and not antibody-mediated  rejection, as diagnostic assays for the presence of antibody-mediated rejection were not routinely performed during the sample collection period.CAV, the leading cause of late morbidity and mortality after heart transplantation, is a complex multifactorial process mediated by both immune and non-immune factors. The diffuse nature of CAV, which usually involves the entire coronary arterial tree a priori plausibility, given their demonstrated utility for diagnosis of acute rejection after kidney transplantation, reflecting common pathways of immune activation rule out the presence of rejection. This assay, with a high PPV, would therefore be complementary by concurrently enabling clinicians to rule in the presence of rejection and additionally predict a risk-read out for AR prior to any clinical graft dysfunction. A strategy that combines both non-invasive tests could therefore enable biopsy avoidance in a larger number of patients than either test alone. The observed gene expression patterns in this study challenge the current paradigm of classifying certain rejection grades, such as Grade 1B, as “mild” and therefore not requiring intensification of immunosuppressive therapy. Further testing in larger patient numbers and prospective clinical trials will be necessary to additionally validate this panel, with the goal of developing a noninvasive and clinically relevant test for diagnosis of cardiac allograft rejection.In conclusion, an internally validated 5-gene classifier panel, from a larger set of 10 genes, has been developed to non-invasively screen for the presence of acute cellular rejection after heart transplantation; the same 10 genes also being diagnostic of acute kidney transplant rejection. The markers studied had


SRY (sex determining region on Y chromosome) located on the short arm of chromosome Y. The differentiation of male external genitalia  occurs between the 9th and 13th weeks of pregnancy and requires adequate concentration of testosterone and the conversion of this to another more potent androgen, dihydrotestosterone, through the action of 5α-reductase in target tissues.Androgen is a generic term usually applied to describe a group of sex steroid hormones. Androgens are responsible for male sex differentiation during embryogenesis at the sixth or seventh week of gestation, triggering the development of the testes and penis in male fetuses, and are directed by the testicular determining factor: the gene This report describes the case of a teenage girl presenting with a male karyotype, and aims to determine the extension of the mutation that affected the AR gene. A Caucasian girl aged 15 was referred to our laboratory for genetic testing due to primary amenorrhea. Physical examination, karyotype testing and molecular analysis of the androgen receptor were critical in making the correct diagnosis of complete androgen insensitivity syndrome.AR gene may cause either complete or partial androgen insensitivity syndrome. The case reported here is consistent with complete androgen insensitivity syndrome, misdiagnosed at birth, and consequently our patient was raised both socially and educationally as a female. It is critical that health care providers understand the importance of properly diagnosing a newborn manifesting ambiguous genitalia. Furthermore, a child with a pseudohermaphrodite phenotype should always undergo adequate endocrine and genetic testing to reach a conclusive diagnosis before gender is assigned and surgical interventions are carried out. Our results show that extreme care must be taken in selecting the genetic tools that are utilized for the diagnosis for androgen insensitivity syndrome.Sex determination and differentiation depend on a cascade of events that begins with the establishment of chromosomal sex at fertilization and ends with sexual maturation at puberty, subsequently leading to fertility. Mutations affecting the  SRY (sex determining region on Y chromosome) located on the short arm of chromosome Y. The differentiation of male external genitalia in the penis, scrotum and penile urethra occurs between the 9th and 13th weeks of pregnancy and requires adequate concentration of testosterone and conversion of this to another more potent androgen, dihydrotestosterone (DHT), through the action of 5α-reductase in target tissues [Androgen is a generic term usually applied to describe a group of sex steroid hormones. Androgens are produced primarily by a male's testes. However, some small amounts are also produced by the ovaries in females and by the adrenal gland in both sexes. Androgens are responsible for male sex differentiation during embryogenesis at the sixth or seventh week of gestation, triggering the development of the testes and penis in male fetuses, and are directed by the testicular determining factor: the gene  tissues .AR gene is a protein-coding gene located at Xq11.2-q12. It spans over 90 kb and codes for a protein that functions as a steroid-hormone-activated transcription factor  is relatively rare. In childhood, the most common clinical presentation is the presence of bilateral inguinal hernias. Individuals not diagnosed during childhood are detected after puberty because of primary amenorrhea. Patients with the complete form of AIS have female external genitalia, an absence or thinning of pubic hair and the absence of a uterus .The AR NTD is relatively long and displays the greatest sequence variability among nuclear receptors. It is very flexible and displays a high degree of intrinsic disorder. In addition, the NTD has a variable number of homopolymeric repeats, the most important of which is a polyglutamine repeat that ranges from eight to 31 repeats in normal individuals, with an average length of 20 base pairs .The DBD is centrally located in the AR and it is the most conserved region within the nuclear receptor family. This region has nine cysteine residues, eight of which are involved in forming two zinc fingers, and a C-terminal extension. The first zinc finger, most proximal to the NTD, determines the specificity of DNA recognition, whereas residues in the second zinc finger are involved in AR dimerization. Two AR monomers in a head-to-head conformation bind as a homodimer to androgen response elements which are direct or indirect repeats of the core consensus 5'-TGTTCT-3' or more complex response elements with diverse arrangements of AREs. The C-terminal extension is important for the three-dimensional structure of the DBD and it plays a role in mediating the AR selectivity of DNA interaction .AR gene [The hinge region has long been considered to be a flexible linker between the DBD and LBD in the AR. More recently, however, this region was shown to be involved in DNA binding as well as AR dimerization. It was suggested that the hinge region also acts to attenuate transcriptional activity of the AR gene .AR gene cause X-linked androgen insensitivity syndrome (AIS) characterized by androgen unresponsiveness, which affects proper male sexual development both at embryogenesis and at puberty. As a genetic disorder, AIS presents problems to affected people and their families, and is a major medical challenge for health providers. This impaired response to androgen results from an incapacity or reduced capacity of the AR to transactivate androgen-responsive genes in target cells, and leads to abnormal differentiation and development of male internal and external genitalia, leading to male pseudo-hermaphroditism [Mutations in the roditism .AR gene result in AIS (OMIN #300068). Depending on the extent of the AR defect, the phenotype can vary. Three forms are classified, complete (CAIS), partial (PAIS), and mild (MAIS). All forms of AIS are X-linked traits inherited as a recessive disorder. Despite a normal male karyotype , individuals affected with CAIS  have female external genitalia, blind vaginas, an absent uterus and female adnexa, female breast development, and abdominal or inguinal testes. Partial androgen insensitivity results in hypospadias and micropenis with gynecomastia [Most mutations on the comastia .in situ hybridization (FISH) and PCR in order to determine the nature and the extent of the mutation that affected the AR gene.The aim of the investigation reported here was to provide a genetic diagnosis of a teenage girl with normal male karyotype using fluorescence A 15-year-old Caucasian girl was referred to a laboratory in Goiânia, Goiás, Brazil, for genetic testing due to primary amenorrhea. Her medical history included removal of an abdominal mass as a newborn. The tissue removed was referred to as an umbilical hernia. Her G-band karyotype revealed a diploid set of chromosomes, including 22 pairs of homologous autosomes and one pair of sex chromosomes, compatible with a 46, XY male chromosome complement.The geneticists at our laboratory concluded that the mass withdrawal from the abdomen of our patient was, in fact, testes, and that our patient had a condition known as cryptorchidism; a reproductive change characterized by a failure of the movement of one or both testes from the abdominal cavity to the scrotum.AR gene and to detect the AR gene, respectively. We prepared a culture of T lymphocytes in RPMI 1640 medium, supplemented with 20% fetal calf serum and 2% of phytohemagglutinin. Metaphasic preparations were made by conventional methodology. The slides for FISH were prepared with a micropipette, with about 15 μL of the material set. Only slides of good quality (in terms of metaphase) were selected by phase contrast microscope, and were subjected to FISH using the LSI Androgen Receptor SpectrumOrange (Xq12) probe . For PCR, primers were used for exons 1, 4, 6, 7 and 8 of AR [We performed PCR and FISH to verify mutations of the exons 1, 4, 6, 7 and 8 of the  8 of AR .In situ hybridization with the LSI AR probe indicated the presence of the gene in all analyzed cells. However, genomic DNA extracted from peripheral blood leukocytes assessed by PCR revealed coding sequence abnormalities for the AR gene, which lacked exons 1 to 7 indicating large deletion spanning the proximal region of the gene. Figure The results reported in this paper underline the fact that great care must be taken when selecting genetic testing tools to be utilized to reach the proper diagnosis for AIS. If a child reaches his or her teenage years undiagnosed due to clinical challenges presented by ambiguous genitalia it further complicates matters, as there could be several genetic events subjacent to that outcome, ranging from total or partial deletion of the gene to point mutation, that efficiently silences the gene and therefore leads to AIS. Here, we report that FISH alone was not able to properly diagnose our patient, despite the proximal deletion within the AR observed on PCR Figure . This reAR gene may cause either complete or partial AIS. The case of our patient reported here is consistent with CAIS, misdiagnosed at birth, and she was consequently raised socially and educationally as a female. It is critical that health care providers understand the importance of properly diagnosing a newborn with ambiguous genitalia. Prompt evaluation of both clinical and genetic findings is crucial to determine proper gender assignment and the detection of life-threatening conditions [Sex determination and differentiation depend on a cascade of events that begins with the establishment of chromosomal sex at fertilization and ends with sexual maturation at puberty subsequently leading to fertility. Mutations affecting the nditions . Furthernditions .The presentation of a patient, and specifically a neonate, with abnormal genital development represents a difficult diagnostic and therapeutic challenge. Referral to a center with experience in the diagnosis and management of disorders of sexual development is advised, where an emphasis should be placed on psychological and genetic counseling ,15.Written informed consent was obtained from the patient's next-of-kin for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.COAM was involved in collating the information, reviewing the literature, and preparation of the manuscript. DMS was involved in collating information regarding the case and getting informed consent from our patient. ADC was involved in the review of literature and revising the manuscript critically. All authors read and approved the final manuscript.