Interactions between serotonergic genotype and amino acid transmitter receptor expression in human alcoholics

Serotonin can modulate the activity of neural reward pathways that are strongly implicated in mediating the effects of chronic alcohol misuse, and its treatment, in human subjects. In previous work and as discussed elsewhere at this meeting, we and others have found consistent differences in the parameters of GABA and glutamate receptors, and the expression of their component subunit transcripts and proteins, in areas of the alcoholic brain that are altered by alcoholism. We did not fi nd clear changes in GABA and glutamate transport function in such samples, but a series of microarray analyses showed consistent upregulation of the presynaptic GABA/betaine transporter SLC6A12. Microarray studies showed no signifi cant differences in the expression of transcripts associated with 5HT transmission; however, only a small number of such elements were present on the arrays. Here we partitioned GABAA and NMDA pharmacology, and subunit mRNA and protein expression, measured in samples of frontal and motor cortex obtained at autopsy from alcoholics without comorbid disease, alcoholics with liver cirrhosis, and controls, according to 5HTTLPR (SLC6A4) and 5HT1B (HTR1B) polymorphisms. We found no effect of these genotypes on the expression of GABAA receptor gene products, but there was a signifi cant mRNA Transcript X Area X Group X 5HTTLPR Interaction with NMDA subunit isoform expression measured by Real Time PCR with GAPDH normalization. Further analysis showed the effect to be selective for alcoholics with cirrhosis, to be most marked in the pathologically vulnerable frontal cortex, and to vary with subunit transcript (F2,76 = 6.545, P = 0.002). NR1 expression was most affected, followed by NR2A, with NR2B expression least altered. Pilot data suggest 5HT1B genotype may also modulate NMDA subunit expression. Interactions between amino acid and serotonin transmission may infl uence susceptibility to alcohol dependence or pathogenesis

The differential impact of oxytocin receptor (OXTR) genotypes on the risk of autism spectrum disorder (ASD) and resulting social communication deficits

Background: Autism spectrum disorder (ASD) is multifactorial and is likely the result of complex interactions between multiple environmental and genetic factors. Recently, it has been suggested that each symptom cluster of the disorder, such as poor social communication, may be mediated by different genetic influences. Genes in the oxytocin pathway, which mediates social behaviours in humans, have been studied with single nucleotide polymorphisms (SNPs) in the oxytocin receptor gene (OXTR) being implicated in ASD. This thesis examines the presence of different oxytocin receptor genotypes, and their associations with ASD and resulting social communication deficits. Methods: The relationship between four OXTR variants and ASD was evaluated in 607 ASD simplex (SPX) families. Cases were compared to their unaffected siblings using a conditional logistic approach. Odds ratios and associated 95 percent confidence intervals were obtained. A second sample of 235 individuals with a diagnosis of ASD was examined to evaluate whether these four OXTR variants were associated with social communication scores on the Autism Diagnostic Interview – Revised (ADI-R). Parameter estimates and associated 95 percent confidence intervals were generated using a linear regression approach. Multiple testing issues were addressed using false discovery adjustments. Results: The rs53576 AG genotype was significantly associated with a lower risk of ASD (OR = 0.707, 95% CI: 0.512-0.975). A single genotype (AG) provided by the rs2254298 marker was found to be significantly associated with higher social communication scores (Parameter estimate = 1.833, SE = 0.762, p = 0.0171). This association was also seen in a Caucasian only and mothers as the respondent samples. No association was significant following false discovery rate adjustments. Conclusion: The findings from these studies provide limited support for the role of OXTR SNPs in ASD, especially in social communication skills. The clinical significance of these associations remains unknown, however, it is likely that these associations do not play a role in the severity of symptoms associated with ASD. Rather, they may be important in the appearance of social deficits due to the rs2254298 markers association with enlarged amygdalas.

Cell cycle-coupled expansion of AR activity promotes cancer progression

The androgen receptor (AR) is required for prostate cancer (PCa) survival and progression, and ablation of AR activity is the first line of therapeutic intervention for disseminated disease. While initially effective, recurrent tumors ultimately arise for which there is no durable cure. Despite the dependence of PCa on AR activity throughout the course of disease, delineation of the AR-dependent transcriptional network that governs disease progression remains elusive, and the function of AR in mitotically active cells is not well understood. Analyzing AR activity as a function of cell cycle revealed an unexpected and highly expanded repertoire of AR-regulated gene networks in actively cycling cells. New AR functions segregated into two major clusters: those that are specific to cycling cells and retained throughout the mitotic cell cycle ('Cell Cycle Common'), versus those that were specifically enriched in a subset of cell cycle phases ('Phase Restricted'). Further analyses identified previously unrecognized AR functions in major pathways associated with clinical PCa progression. Illustrating the impact of these unmasked AR-driven pathways, dihydroceramide desaturase 1 was identified as an AR-regulated gene in mitotically active cells that promoted pro-metastatic phenotypes, and in advanced PCa proved to be highly associated with development of metastases, recurrence after therapeutic intervention and reduced overall survival. Taken together, these findings delineate AR function in mitotically active tumor cells, thus providing critical insight into the molecular basis by which AR promotes development of lethal PCa and nominate new avenues for therapeutic intervention.

Calcium Channel Blocker Use and Risk of Prostate Cancer by TMPRSS2:ERG Gene Fusion Status

BACKGROUND: Calcium channel blockers (CCBs) may affect prostate cancer (PCa) growth by various mechanisms including those related to androgens. The fusion of the androgen-regulated gene TMPRSS2 and the oncogene ERG (TMPRSS2:ERG or T2E) is common in PCa, and prostate tumors that harbor the gene fusion are believed to represent a distinct disease subtype. We studied the association of CCB use with the risk of PCa, and molecular subtypes of PCa defined by T2E status.

METHODS: Participants were residents of King County, Washington, recruited for population-based case-control studies (1993-1996 or 2002-2005). Tumor T2E status was determined by fluorescence in situ hybridization using tumor tissue specimens from radical prostatectomy. Detailed information on use of CCBs and other variables was obtained through in-person interviews. Binomial and polytomous logistic regression were used to generate odds ratios (ORs) and 95% confidence intervals (CIs).

RESULTS: The study included 1,747 PCa patients and 1,635 age-matched controls. A subset of 563 patients treated with radical prostatectomy had T2E status determined, of which 295 were T2E positive (52%). Use of CCBs (ever vs. never) was not associated with overall PCa risk. However, among European-American men, users had a reduced risk of higher-grade PCa (Gleason scores ≥7: adjusted OR = 0.64; 95% CI: 0.44-0.95). Further, use of CCBs was associated with a reduced risk of T2E positive PCa (adjusted OR = 0.38; 95% CI: 0.19-0.78), but was not associated with T2E negative PCa.

CONCLUSIONS: This study found suggestive evidence that use of CCBs is associated with reduced relative risks for higher Gleason score and T2E positive PCa. Future studies of PCa etiology should consider etiologic heterogeneity as PCa subtypes may develop through different causal pathways. 

TRIM24 Is an Oncogenic Transcriptional Activator in Prostate Cancer.

Androgen receptor (AR) signaling is a key driver of prostate cancer (PC). While androgen-deprivation therapy is transiently effective in advanced disease, tumors often progress to a lethal castration-resistant state (CRPC). We show that recurrent PC-driver mutations in speckle-type POZ protein (SPOP) stabilize the TRIM24 protein, which promotes proliferation under low androgen conditions. TRIM24 augments AR signaling, and AR and TRIM24 co-activated genes are significantly upregulated in CRPC. Expression of TRIM24 protein increases from primary PC to CRPC, and both TRIM24 protein levels and the AR/TRIM24 gene signature predict disease recurrence. Analyses in CRPC cells reveal that the TRIM24 bromodomain and the AR-interacting motif are essential to support proliferation. These data provide a rationale for therapeutic TRIM24 targeting in SPOP mutant and CRPC patients.

Epigenetic and oncogenic regulation of SLC16A7 (MCT2) results in protein over-expression, impacting on signalling and cellular phenotypes in prostate cancer

Monocarboxylate Transporter 2 (MCT2) is a major pyruvate transporter encoded by the SLC16A7 gene. Recent studies pointed to a consistent overexpression of MCT2 in prostate cancer (PCa) suggesting MCT2 as a putative biomarker and molecular target. Despite the importance of this observation the mechanisms involved in MCT2 regulation are unknown. Through an integrative analysis we have discovered that selective demethylation of an internal SLC16A7/MCT2 promoter is a recurrent event in independent PCa cohorts. This demethylation is associated with expression of isoforms differing only in 5'-UTR translational control motifs, providing one contributing mechanism for MCT2 protein overexpression in PCa. Genes co-expressed with SLC16A7/MCT2 also clustered in oncogenic-related pathways and effectors of these signalling pathways were found to bind at the SLC16A7/MCT2 gene locus. Finally, MCT2 knock-down attenuated the growth of PCa cells. The present study unveils an unexpected epigenetic regulation of SLC16A7/MCT2 isoforms and identifies a link between SLC16A7/MCT2, Androgen Receptor (AR), ETS-related gene (ERG) and other oncogenic pathways in PCa. These results underscore the importance of combining data from epigenetic, transcriptomic and protein level changes to allow more comprehensive insights into the mechanisms underlying protein expression, that in our case provide additional weight to MCT2 as a candidate biomarker and molecular target in PCa.

Combining molecular alterations and functional imaging in metastatic castration resistant prostate cancer treated with taxanes

The treatment of metastatic castration-resistant prostate cancer (mCRPC) is currently characterized by several drugs with different mechanisms of action, such as new generation hormonal agents (abiraterone, enzalutamide), chemotherapy (docetaxel, cabazitaxel), PARP inhibitors (olaparib) and radiometabolic therapies (radium-223, LuPSMA). There is an urgent need to identify biomarkers to guide personalized therapy in mCRPC. In recent years, the status of androgen receptor (AR) gene detected in liquid biopsy has been associated with outcomes in patients treated with abiraterone or enzalutamide. More recently, plasma tumor DNA (ptDNA) and its changes during treatment have been identified as early indicators of response to anticancer treatments. Recent works also suggested a potential role of tumor-related metabolic parameters of 18Fluoro-Choline Positron Emission Tomography (F18CH-PET)-computed tomography (CT) as a prognostic tool in mCRCP. Other clinical features, such as the presence of visceral metastases, have been correlated with outcome in mCRPC patients. Recent studies conducted by our research group have designed and validated a prognostic model based on the combination of molecular characteristics (ptDNA levels), metabolic features found in basal FCH PET scans (metabolic tumor volume values, MTV), clinical parameters (absence or presence of visceral metastases), and laboratory tests (serum lactate dehydrogenase levels, LDH). Within this PhD project, 30 patients affected by mCRPC, pre-treated with abiraterone or enzalutamide, candidate for taxane-based treatments (docetaxel or cabazitaxel), have been prospectively evaluated. The prognostic model previously described was applied to this population, to interrogate its prognostic power in a more advanced cohort of patients, resulting in a further external validation of the tool.

Analysis Of The Contribution Of Immunologically-detectable Her2, Steroid Receptors And Of The Triple-negative" Tumor Status To Disease-free And Overall Survival Of Women With Epithelial Ovarian Cancer."

We assessed associations between steroid receptors including: estrogen-alpha, estrogen-beta, androgen receptor, progesterone receptor, the HER2 status and triple-negative epithelial ovarian cancer (ERα-/PR-/HER2-; TNEOC) status and survival in women with epithelial ovarian cancer. The study included 152 women with primary epithelial ovarian cancer. The status of steroid receptor and HER2 was determined by immunohistochemistry. Disease-free and overall survival were calculated and compared with steroid receptor and HER2 status as well as clinicopathological features using the Cox Proportional Hazards model. A mean follow-up period of 43.6 months (interquartile range=41.4 months) was achieved where 44% of patients had serous tumor, followed by mucinous (23%), endometrioid (9%), mixed (9%), undifferentiated (8.5%) and clear cell tumors (5.3%). ER-alpha staining was associated with grade II-III tumors. Progesterone receptor staining was positively associated with a Body Mass Index≥25. Androgen receptor positivity was higher in serous tumors. In stand-alone analysis of receptor contribution to survival, estrogen-alpha positivity was associated with greater disease-free survival. However, there was no significant association between steroid receptor expression, HER2 status, or TNEOC status, and overall survival. Although estrogen-alpha, androgen receptor, progesterone receptor and the HER2 status were associated with key clinical features of the women and pathological characteristics of the tumors, these associations were not implicated in survival. Interestingly, women with TNEOC seem to fare the same way as their counterparts with non-TNEOC.

A simplified minimal residual disease polymerase chain reaction method at early treatment points can stratify children with acute lymphoblastic leukemia into good and poor outcome groups

Background Minimal residual disease is an important independent prognostic factor in childhood acute lymphoblastic leukemia. The classical detection methods such as multiparameter flow cytometry and real-time quantitative polymerase chain reaction analysis are expensive, time-consuming and complex, and require considerable technical expertise. Design and Methods We analyzed 229 consecutive children with acute lymphoblastic leukemia treated according to the GBTLI-99 protocol at three different Brazilian centers. Minimal residual disease was analyzed in bone marrow samples at diagnosis and on days 14 and 28 by conventional homo/heteroduplex polymerase chain reaction using a simplified approach with consensus primers for IG and TCR gene rearrangements. Results At least one marker was detected by polymerase chain reaction in 96.4%, of the patients. By combining the minimal residual disease results obtained on days 14 and 28, three different prognostic groups were identified: minimal residual disease negative on days 14 and 28, positive on day 14/negative on day 28, and positive on both. Five-year event-free survival rates were 85%, 75.6%,, and 27.8%, respectively (p<0.0001). The same pattern of stratification held true for the group of intensively treated children. When analyzed in other subgroups of patients such as those at standard and high risk at diagnosis, those with positive B-derived CD10, patients positive for the TEL/AML1 transcript, and patients in morphological remission on a day 28 marrow, the event-free survival rate was found to be significantly lower in patients with positive minimal residual disease on day 28. Multivariate analysis demonstrated that the detection of minimal residual disease on day 28 is the most significant prognostic factor. Conclusions This simplified strategy for detection of minimal residual disease was feasible, reproducible, cheaper and simpler when compared with other methods, and allowed powerful discrimination between children with acute lymphoblastic leukemia with a good and poor outcome.

Melatonin reduces LH, 17 beta-estradiol and induces differential regulation of sex steroid receptors in reproductive tissues during rat ovulation

Background: Melatonin is associated with direct or indirect actions upon female reproductive function. However, its effects on sex hormones and steroid receptors during ovulation are not clearly defined. This study aimed to verify whether exposure to long-term melatonin is able to cause reproductive hormonal disturbances as well as their role on sex steroid receptors in the rat ovary, oviduct and uterus during ovulation. Methods: Twenty-four adult Wistar rats, 60 days old (+/-250 g) were randomly divided into two groups. Control group (Co): received 0.9% NaCl 0.3 mL + 95% ethanol 0.04 mL as vehicle; Melatonin-treated group (MEL): received vehicle + melatonin [ 100 mu g/100 g BW/day] both intraperitoneally during 60 days. All animals were euthanized by decapitation during the morning estrus at 4 a. m. Results: Melatonin significantly reduced the plasma levels of LH and 17 beta-estradiol, while urinary 6-sulfatoximelatonin (STM) was increased at the morning estrus. In addition, melatonin promoted differential regulation of the estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR) and melatonin receptor (MTR) along the reproductive tissues. In ovary, melatonin induced a down-regulation of ER-alpha and PRB levels. Conversely, it was observed that PRA and MT1R were up-regulated. In oviduct, AR and ER-alpha levels were down-regulated, in contrast to high expression of both PRA and PRB. Finally, the ER-beta and PRB levels were down-regulated in uterus tissue and only MT1R was up-regulated. Conclusions: We suggest that melatonin partially suppress the hypothalamus-pituitary-ovarian axis, in addition, it induces differential regulation of sex steroid receptors in the ovary, oviduct and uterus during ovulation.

Exercise training delays cardiac dysfunction and prevents calcium handling abnormalities in sympathetic hyperactivity-induced heart failure mice

Exercise training (ET) is a coadjuvant therapy in preventive cardiology. It delays cardiac dysfunction and exercise intolerance in heart failure (HF); however, the molecular mechanisms underlying its cardioprotection are poorly understood. We tested the hypothesis that ET would prevent Ca2+ handling abnormalities and ventricular dysfunction in sympathetic hyperactivity-induced HF mice. A cohort of male wildtype (WT) and congenic (alpha 2A/alpha 2C)-adrenoceptor knockout ((alpha 2A/alpha 2C)ARKO) mice with C57BL6/J genetic background (3-5 mo of age) were randomly assigned into untrained and exercise-trained groups. ET consisted of 8-wk swimming session, 60 min, 5 days/wk. Fractional shortening (FS) was assessed by two-dimensional guided M-mode echocardiography. The protein expression of ryanodine receptor (RyR), phospho-Ser(2809)-RyR, sarcoplasmic reticulum Ca2+ ATPase (SERCA2), Na+/Ca2+ exchanger (NCX), phospholamban (PLN), phospho-Ser(16)-PLN, and phospho-Thr(17)-PLN were analyzed by Western blotting. At 3 mo of age, no significant difference in FS and exercise tolerance was observed between WT and (alpha 2A/alpha 2C)ARKO mice. At 5 mo, when cardiac dysfunction is associated with lung edema and increased plasma norepinephrine levels, (alpha 2A/alpha 2C)ARKO mice presented reduced FS paralleled by decreased SERCA2 (26%) and NCX (34%). Conversely, (alpha 2A/alpha 2C)ARKO mice displayed increased phospho-Ser(16)-PLN (76%) and phospho-Ser(2809)-RyR (49%). ET in (alpha 2A/alpha 2C)ARKO mice prevented exercise intolerance, ventricular dysfunction, and decreased plasma norepinephrine. ET significantly increased the expression of SERCA2 (58%) and phospho-Ser(16)-PLN (30%) while it restored the expression of phospho-Ser(2809)-RyR to WT levels. Collectively, we provide evidence that improved net balance of Ca2+ handling proteins paralleled by a decreased sympathetic activity on ET are, at least in part, compensatory mechanisms against deteriorating ventricular function in HF.

Effects of methylmercury on male reproductive functions in Wistar rats

In this study we investigated the effects of subacute exposure to methylmercury (MeHg) on male reproductive functions in rats by means of determination of alterations in structural and functional parameters. Adult male Wistar rats received 0, 0.5, 1.0 or 3.0 mg/kg/body weight/day orally, daily MeHg for 14 days. Sperm motility, the relative sperm count and transit time in the caput/corpus epididymis, were all reduced at all doses. The lowest dose increased the number of sperm head abnormalities; daily sperm production was elevated at the intermediate dose; while at the highest dose there was a decrease in serum testosterone levels and a rise in mercury (Hg) content in reproductive organs, liver and kidneys. In conclusion, MeHg exposure produced damages on male reproductive functions which may be attributed, at least in part, to the reduction in serum testosterone levels. These consequences could potentially result in infertility in rats. (C) 2011 Elsevier Inc. All rights reserved.

Hepatocellular carcinoma

Detailed analyses of chromosomal damage in hepatocellular carcinoma have confirmed the results of previous studies that identified regions of significant loss. In addition, these studies examined the clinicopathological correlates of this damage, identified new sites for future investigation, and provided evidence of interactions between genes, The insulin-like growth factor II receptor gene is a target for inactivation through chromosomal loss and mutation, with loss also occurring in the cirrhotic liver. The insulin-like growth factor II receptor gene plays a central role in coordinating the competing actions of insulin-like growth factor and transforming growth factor-beta on cell proliferation. Our understanding of the changes in these growth factor pathways helps explain the apparent increase in risk of hepatocellular carcinoma in diabetic patients and the potential use of urinary transforming growth factor-beta in screening tests. Vaccination for hepatitis B in Taiwan has had a significant effect on the incidence of childhood hepatocellular carcinoma. Universal vaccination should result in a major reduction in the incidence of hepatocellular carcinoma worldwide.

Plasminogen activator system in osteoclasts

To determine which genes of the plasminogen activator (PA) system were expressed in osteoclasts, RNA extracted from microisolated mouse osteoclasts was used as template for reverse transcribed polymerase chain reaction (RT-PCR) with gene-specific primer pairs, Using this approach, the expression of RNAs for tissue-type plasminogen activator, urokinase-type plasminogen activator, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, protease nexin, and urokinase receptor isoform 1 (uPAR1) were detected in mouse osteoclasts. The expression of uPAR RNA in osteoclasts was confirmed by in situ hybridization with a uPAR1 probe, RNA encoding the uPAR isoform 2 was not detected in mouse osteoclasts, but a novel unspliced uPAR RNA variant was detected in these cells, The novel uPAR variant and uPAR1 RNA were also detected in mouse calvarial osteoblasts, kidney, muscle, and the mouse macrophage cell line J774A.1 by RT-PCR The presence of RNAs for most of the components of the PA system in osteoclasts suggests that it may have a functional role in this cell type.

Proposal for the interaction of non-conventional partial agonists and catecholamines with the 'putative beta(4)-adrenoceptor' in mammalian heart

1. Evidence for a 'putative beta(4)-adrenoceptor' originated over 20 years ago when cardiostimulant effects were observed to nonconventional partial agonists, These agonists were originally described as beta(1)- and beta(2)-adrenoceptor antagonists; however, they cause cardiostimulant effects at much higher concentrations than those required to block beta(1)- and beta(2)-adrenoceptors. Cardiostimulant effects of non-conventional partial agonists have been observed in mouse, rat, guinea-pig, cat, ferret and human heart tissues, 2. The receptor is expressed in several heart regions, including the sinoatrial node, atrium and ventricle, 3. The receptor is resistant to blockade by most antagonists that possess high affinity for beta(1)- and beta(2)- adrenoceptors, but is blocked with moderate affinity by (-)-bupranolol and CGP 20712A. 4. The receptor is pharmacologically distinct from the beta(3)-adrenoceptor. Micromolar concentrations of beta(3)-adrenoceptor agonists have no agonist or blocking activity, The receptor is also resistant to blockade by a beta(3)-adrenoceptor-selective antagonist. 5. The receptor mediates increases in cAMP levels and cAMP-dependent protein kinase (PK) A activity in cardiac tissues. Phosphodiesterase inhibition potentiates the positive chronotropic and inotropic effects of non-conventional partial agonists. 6. The receptor mediates hastening of atrial and ventricular relaxation, which is consistent with involvement of a cAMP-dependent pathway. 7. The non-conventional partial agonist (-)-[H-3]-CGP 12177A labels the cardiac putative beta(4)-adrenoceptor, Non-conventional partial agonists compete for binding with affinities that are closely similar to their agonist potencies, Catecholamines compete for binding in a stereoselective manner with a rank order of affinity of (-)-R0363 > (-)-isoprenaline > (-)-noradrenaline greater than or equal to (-)-adrenaline much greater than (-)-isoprenaline, suggesting that catecholamines can interact with the receptor. 8. The putative beta(4)-adrenoceptor appears to be coupled to the G(s)-adenylyl cyclase system, which could serve as a guide to its future cloning, Activation of the receptor may plausibly improve diastolic function but could also mediate arrhythmias.