bluuebunny/crossref_metadata_2025 huggingface dataset:
Estimation of genotype relative risks from pedigree data by retrospective likelihoods
Authors: Schaid Daniel J. , McDonnell Shannon K. , Riska Shaun M. , Carlson Erin E. , Thibodeau Stephen N.
Published: December 2009

Pedigrees collected for linkage studies are a valuable resource that could be used to estimate genetic relative risks (RRs) for genetic variants recently discovered in case‐control genome wide association studies. To estimate RRs from highly ascertained pedigrees, a pedigree “retrospective likelihood” can be used, which adjusts for ascertainment by conditioning on the phenotypes of pedigree members. We explore a variety of approaches to compute the retrospective likelihood, and illustrate a Newton‐Raphson method that is computationally efficient particularly for single nucleotide polymorphisms (SNPs) modeled as log‐additive effect of alleles on the RR. We also illustrate, by simulations, that a naïve “composite likelihood” method that can lead to biased RR estimates, mainly by not conditioning on the ascertainment process—or as we propose—the disease status of all pedigree members. Applications of the retrospective likelihood to pedigrees collected for a prostate cancer linkage study and recently reported risk‐SNPs illustrate the utility of our methods, with results showing that the RRs estimated from the highly ascertained pedigrees are consistent with odds ratios estimated in case‐control studies. We also evaluate the potential impact of residual correlations of disease risk among family members due to shared unmeasured risk factors (genetic or environmental) by allowing for a random baseline risk parameter. When modeling only the affected family members in our data, there was little evidence for heterogeneity in baseline risks across families. Genet. Epidemiol. 34: 287–298, 2010. © 2009 Wiley‐Liss, Inc.
Case‐only gene‐environment interaction studies: when does association imply mechanistic interaction?
Authors: VanderWeele Tyler J. , Hernández‐Díaz Sonia , Hernán Miguel A.
Published: December 2009

Case‐only studies are often used to identify interactions between a genetic factor and an environmental factor under the assumption both factors are independent in the population. However, interpreting a statistical association between the genetic and the environmental factors among the cases, as evidence of a mechanistic gene‐environment interaction, is not always warranted. Using a mechanistic approach based on the sufficient cause framework, we show association amongst cases can arise between the genetic and environmental factors when there is in fact no mechanistic gene‐environment interaction. However, when it can be assumed the genetic and environmental factors themselves can never prevent the outcome, we show a positive association amongst cases implies a mechanistic gene‐environment interaction. Without this assumption that the effects of the two factors are never preventive, a multiplicative interaction greater than two is needed to conclude the presence of a mechanistic interaction. We furthermore show these tests for mechanistic interaction can be extended to scenarios in which the genetic and environmental factors are negatively associated in the population rather than independent. Genet. Epidemiol. 34: 327–334, 2010. © 2009 Wiley‐Liss, Inc.
A powerful approach to sub‐phenotype analysis in population‐based genetic association studies
Authors: Morris Andrew P. , Lindgren Cecilia M. , Zeggini Eleftheria , Timpson Nicholas J. , Frayling Timothy M. , Hattersley Andrew T. , McCarthy Mark I.
Published: December 2009

The ultimate goal of genome‐wide association (GWA) studies is to identify genetic variants contributing effects to complex phenotypes in order to improve our understanding of the biological architecture underlying the trait. One approach to allow us to meet this challenge is to consider more refined sub‐phenotypes of disease, defined by pattern of symptoms, for example, which may be physiologically distinct, and thus may have different underlying genetic causes. The disadvantage of sub‐phenotype analysis is that large disease cohorts are sub‐divided into smaller case categories, thus reducing power to detect association. To address this issue, we have developed a novel test of association within a multinomial regression modeling framework, allowing for heterogeneity of genetic effects between sub‐phenotypes. The modeling framework is extremely flexible, and can be generalized to any number of distinct sub‐phenotypes. Simulations demonstrate the power of the multinomial regression‐based analysis over existing methods when genetic effects differ between sub‐phenotypes, with minimal loss of power when these effects are homogenous for the unified phenotype. Application of the multinomial regression analysis to a genome‐wide association study of type 2 diabetes, with cases categorized according to body mass index, highlights previously recognized differential mechanisms underlying obese and non‐obese forms of the disease, and provides evidence of a potential novel association that warrants follow‐up in independent replication cohorts. Genet. Epidemiol. 34: 335–343, 2010.  © 2009 Wiley‐Liss, Inc.
Alterations in cortical activity of male methamphetamine abusers performing an empathy task: fMRI study
Authors: Kim Yang‐Tae , Lee Jae‐Jun , Song Hui‐Jin , Kim Joo‐Hyun , Kwon Do‐Hoon , Kim Myoung‐Nam , Yoo Done‐Sik , Lee Hui Joong , Kim Hye‐Jung , Chang Yongmin
Published: December 2009

Objectives

We investigate possible differences in neural correlates of empathy processing between abstinent methamphetamine (MA) abusers and healthy subjects using functional magnetic resonance imaging (fMRI).

Methods

Nineteen abstinent MA abusers (mean age of 36.06 years, range 31–52 years) and 19 healthy subjects (mean age of 37.05 years, range 33–42 years) participated in this study. A visual fMRI activation paradigm was used, comprising a series of cartoons, each depicting a short story. There were two categories of stories: empathy (Empathy) and Physical causality (Physical). fMRI images were acquired using a 3.0 T whole‐body scanner. All fMRI data were analyzed using MATLAB v. 7.2 and SPM5.

Results

Both MA subjects and controls exhibited activation in the dorsomedial prefrontal cortex. Despite this similarity in activation patterns, we found that the two groups differed in the activation of several cortical regions associated with the processing of empathy information. Hypoactivations of the orbitofrontal cortex, temporal poles, and hippocampus in MA abusers relative to healthy subjects suggests that the ability of empathic response could be compromised in abstinent MA abusers (p < 0.05, corrected for a small volume).

Conclusions

Functional impairments in the empathic neural network caused by MA may contribute to the misunderstanding of others and to the erosion of social interactions in MA abusers. Copyright © 2009 John Wiley & Sons, Ltd.
Effect of metabolic blockade on the psychoactive effects of dextromethorphan
Authors: Zawertailo Laurie A. , Tyndale Rachel F. , Busto U. , Sellers Edward M.
Published: December 2009

Objective

Variation in the activity of cytochrome P450 2D6 (CYP2D6) affects the pharmacokinetics and effectiveness of dextromethorphan (DM), because it controls the production of dextrorphan, an active metabolite, with higher affinity for the NMDA receptor than the parent compound. This study examined whether pharmacological inhibition of CYP2D6 activity with quinidine would mimic the genetic mutation and thus also alter the psychoactive effects of DM.

Methods

In a single‐blind, within‐subjects study, eight healthy volunteers (all homozygous for the wild type allele for CYP2D6) received placebo and varying doses of DM, both with and without quinidine pre‐treatment. Pharmacokinetic and pharmacodynamic measures were assessed at baseline and every hour post‐drug for 6 h.

Results

Compared to the no quinidine condition, quinidine pre‐treatment decreased the area under the dose–response curve on subjective measures of positively reinforcing effects (e.g., euphoria,p < 0.04; drug liking,p < 0.05), and was significantly greater for measures of dysphoria (e.g., unpleasantness,p < 0.02). These changes corresponded to increased DM and decreased dextrorphan plasma concentrations.

Conclusions

Compared to DM alone, quinidine pre‐treatment inhibited DM metabolism and changed its subjective effects, demonstrating that the psychoactive properties of DM are a function of drug metabolism. These results demonstrate the relationship between CYP2D6 activity, plasma drug levels, and psychoactive drug effects, and have implications for both the abuse liability and therapeutic utility of DM. Copyright © 2009 John Wiley & Sons, Ltd.
Missense polymorphisms in three oxidative‐stress enzymes (GSTP1, SOD2, and GPX1) and dyskinesias in Russian psychiatric inpatients from Siberia
Authors: Al Hadithy A. F. Y. , Ivanova S. A. , Pechlivanoglou P. , Wilffert B. , Semke A. , Fedorenko O. , Kornetova E. , Ryadovaya L. , Brouwers J. R. B. J. , Loonen A. J. M.
Published: December 2009

Neuronal degeneration due to oxidative stress (OS) has been proposed as a mechanism for tardive dyskinesia (TD) pathogenesis. Cellular defense mechanisms against OS may involve detoxification enzymes (e.g., glutathione peroxidase‐1, GPX1; superoxide dismutase‐2, SOD2 [also commonly known as MnSOD]; and glutathione S‐transferase P1, GSTP1). Several pharmacogenetic studies have examined TD and OS in different ethnic groups, but not in Russians. Here we report the association between orofaciolingual (TDof) and limb‐truncal dyskinesias (TDlt) and polymorphisms of GSTP1 (Ile105Val), MnSOD (Ala‐9Val), and GPX1 (Pro197Leu) genes in 146 Russian inpatients from Siberia. We applied AIMS instrument to rate dyskinesias. Two‐part model analyses, logistic and multivariate parametric regressions were applied to assess the effects of different variables (e.g., genotype, age, gender, and medication use). Our analyses do not suggest that Pro197Leu (GPX1) is associated with TD. However, our analyses suggest that the 105Val‐allele of Ile105Val (GSTP1) may be associated with a lower risk and a severity of TDof and TDlt and that Ile105Val pharmacogenetics may be different in Slavonic Caucasians from that in American Caucasians. Furthermore, we find evidence for an association between Ala‐9Val (MnSOD) and TDof, but not TDlt. Subject to further replication, our findings extend the available knowledge on the pharmacogenetics of TD and oxidative stress. Copyright © 2009 John Wiley & Sons, Ltd.
Analysis of anti‐proliferative and chemosensitizing effects of sunitinib on human esophagogastric cancer cells: Synergistic interaction with vandetanib via inhibition of multi‐receptor tyrosine kinase pathways
Authors: Lyros Orestis , Mueller Annett , Heidel Florian , Schimanski Carl C. , Gockel Ines , Galle Peter R. , Lang Hauke , Moehler Markus
Published: December 2009

The receptor tyrosine kinases (RTKs), epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 1‐3 (VEGFR1‐3), are frequently expressed in gastric cancer and are putative therapeutic targets in this disease. We have investigated the anti‐proliferative and chemosensitizing properties of the multitargeted small‐molecule RTK inhibitors sunitinib and vandetanib in a panel of 4 human gastric and esophageal cancer cell lines. In the 1st instance, the expression of potential targets of these small‐molecule inhibitors was examined by reverse transcriptase‐polymerase chain reaction, western blotting, and flow cytometry. EGFR mRNA and protein was detected in all cases, with VEGFR2 expression noted in all but 1 line. Both EGF and VEGF were shown to stimulate tumor cell growth, and both sunitinib and vandetanib were found to be associated with significant dose‐dependent inhibition of proliferation and an enhancement of apoptosis, as determined by MTT and propidium iodide/Annexin V labeling assays, respectively. The addition of sunitinib to VEGF‐stimulated NCI‐N87 cells was associated with a reduction in MAPK phosphorylation (pMAPK) but not Akt phosphorylation (pAkt), whereas the addition of vandetanib was associated with reductions in both VEGF‐ and EGF‐mediated VEGFR2 phosphorylation, pMAPK and pAkt. Co‐administration of sunitinib significantly enhanced the sensitivity of MKN‐45 cells to cisplatin and irinotecan. In addition, vandetanib synergistically enhanced the sunitinib‐associated inhibition of gastric cancer cell growth. In conclusion, these preliminary data confirm the importance of EGFR and VEGFR signaling in gastric cancer and suggest that the simultaneous inhibition of RTK‐pathways through sunitinib and vandetanib may provide therapeutic benefit in this disease.
Serum carotenoid, tocopherol and retinol concentrations and breast cancer risk in the E3N‐EPIC study
Authors: Maillard Virginie , Kuriki Kiyonori , Lefebvre Benoît , Boutron‐Ruault Marie‐Christine , Lenoir Gilbert M. , Joulin Virginie , Clavel‐Chapelon Françoise , Chajès Véronique
Published: December 2009

Evidence of a protective effect of fruit and vegetable intake on breast cancer risk is inconsistent. Epidemiologic cohort studies based on blood carotenoid intakes as biomarkers of consumption of fruits and vegetable in individuals are still scare and findings are discrepant. The study population included women in the E3N Study, the large French component of the European Prospective Investigation into Cancer and Nutrition (EPIC). During an average of 7 years follow‐up, 366 cases of incident invasive breast cancer (84 premenopausal women and 282 postmenopausal women) among 19,934 women who completed a dietary questionnaire and had available blood samples at baseline (1995–1998) were included in the study. Controls were randomly matched on age, menopausal status at blood collection, fasting status at blood collection, date and collection center. Serum carotenoids, tocopherols and retinol concentrations were assessed by high pressure liquid chromatography. Odds ratios for breast cancer risk adjusted for established breast cancer risk factors were calculated by quintile of serum micronutrient concentrations. No significant associations between breast cancer risk and serum carotenoids (highest versus lowest quintile, odds ratio (OR) = 0.74, 95% confidence interval (CI) = 0.47–1.16, p for trend 0.38), tocopherols (OR = 0.68, 95% CI = 0.41–1.10, p for trend 0.26) and retinol (OR = 0.85, 95% CI = 0.53–1.35, p for trend 0.34) were found. Our findings did not support the hypothesis that lipophilic antioxidant micronutrients found in fruits and vegetables protect against breast cancer, at least in postmenopausal women.
Association between antibiotic use and risk of prostate cancer
Authors: Tamim Hani M. , Hajeer Ali H. , Boivin Jean‐François , Collet Jean‐Paul
Published: December 2009

The association between antibiotics and risk of cancer has been addressed in different studies, most of which were addressing breast cancer. The objective of this study was to assess the association between antibiotics use and risk of prostate cancer. We carried out a population‐based case‐control study using data from Saskatchewan Health administrative databases (Canada) between the years 1981 and 2000. Cases identified by the Saskatchewan Cancer Agency were matched to 4 controls, using incidence density sampling. The effect of dosage and timing of antibiotic use, over a minimum of 15 years before diagnosis, on prostate cancer risk was assessed. Number of prescriptions and number of tablets were used as exposure definitions. Moreover, the effect of different classes of antibiotics on prostate cancer was also studied. A total of 4,052 prostate cancer cases and 16,208 matched controls were included in this study. Antibiotics exposure (number of prescriptions) during the period of 1–15 years in the past was significantly associated with an increased risk of prostate cancer; RR = 1.69, 2.61, 2.71, and 2.83 for the 4 quartiles, respectively, p‐trend = 0.0001. When number of units was taken as the exposure definition, similar results were found. We did not find any effect of the timing or class of antibiotic exposure on prostate cancer risk. We found a dose‐dependent association between antibiotics exposure up to 15 years in the past and risk of prostate cancer. However, the lack of temporal trends and the absence of class specific effects suggest a noncausal relationship.
Transient depletion of CD4+ T cells augments IL‐21‐based immunotherapy of disseminated neuroblastoma in syngeneic mice
Authors: Croce Michela , Corrias Maria Valeria , Orengo Anna Maria , Brizzolara Antonella , Carlini Barbara , Borghi Martina , Rigo Valentina , Pistoia Vito , Ferrini Silvano
Published: December 2009

IL‐21 is a member of the IL‐2 cytokine family, produced by CD4+ T cells. We previously showed that immunotherapy (IT) with IL‐21‐transduced neuroblastoma cells (Neuro2a/IL‐21) cured 33% of syngeneic mice bearing systemic NB. Here, we studied whether the removal of Treg cells could potentiate the therapeutic efficacy of Neuro2a/IL‐21 vaccine. The administration of anti‐CD25 mAb, which targets Treg cells, slightly potentiated the effect of vaccine IT (50% cure rate), but anti‐CD4 mAb had a more potent effect leading to 80% cure rate. Anti‐CD25 mAb, indeed, only partially depleted CD4+CD25+FoxP3+ Treg cells, whereas anti‐CD4 mAb was more effective in this respect, leading to 90% depletion of Treg cells. In mice receiving vaccine+anti‐CD4 mAb, which developed systemic immunity to NB, CD4+ T cells counts completely recovered in 90 days. Depletion of CD8+ T cells abrogated the effect of the combined IT, indicating a predominant role of these cells in driving the immune response. In addition, CD8+ T cells from cured mice coinjected with Neuro2a/parental cells (pc) in NOD‐SCID mice completely inhibited tumor growth. Spleen cells from mice receiving Neuro2a/IL‐21 vaccination showed increased expression of IFN‐alpha2, ‐beta1 and ‐gamma mRNA. Moreover, mice receiving vaccine therapy alone or vaccine+anti‐CD4 mAb showed increased IFN‐gamma serum levels and IFN‐gamma‐producing CD8+ T cells were found in spleen cells. In conclusion, anti‐CD4 mAb potentiated IL‐21‐based IT by removing Treg cells and/or their precursors and other potentially immune‐suppressive CD4+ cell subsets, thus allowing the development of an IL‐21‐driven CD8+ T cell response, which mediates NB rejection.
Detoxification of aristolochic acid I by O‐demethylation: Less nephrotoxicity and genotoxicity of aristolochic acid Ia in rodents
Authors: Shibutani Shinya , Bonala Radha R. , Rosenquist Thomas , Rieger Robert , Suzuki Naomi , Johnson Francis , Miller Frederick , Grollman Arthur P.
Published: December 2009

Ingestion of aristolochic acids (AA) contained in herbal remedies results in aristolochic acid nephropathy (AAN), which is characterized by chronic renal failure, tubulointerstitial fibrosis and urothelial cancer. AA I and AA II, primary components in AA, have similar genotoxic potential, whereas only AA I shows severe renal toxicity in rodents. AA I is demethylated to form 8‐hydroxy‐aristolochic acid I (AA Ia) as a major metabolite. However, the nephrotoxicity and genotoxicity of AA Ia has not yet been determined. AA Ia was isolated from urine collected from rats treated with AA I and characterized by NMR and mass spectrometry. The purified AA Ia was administered intraperitoneally to C3H/He male mice for 9 days and its toxicity was compared with AA I. Using 32P‐postlabeling/polyacrylamide gel electrophoresis, the level of AA Ia‐derived DNA adducts in renal cortex was ∼70–110 times lower than that observed with AA I, indicating that AA Ia has only a limited genotoxicity. Supporting this result, when calf thymus DNA was reacted with AA Ia in a buffer containing zinc dust, the formation of AA Ia‐DNA adducts was two‐orders of magnitude lower than that of AA I. Histopathologic analysis revealed that unlike AA I, no significant changes were detected in the renal cortex of mice treated with AA Ia. Therefore, the contribution of AA Ia to renal toxicity is minimum. We conclude the metabolic pathway of converting AA I to AA Ia functions as the detoxification of AA I.
Prevalence of type‐specific human papillomavirus in endocervical, upper and lower vaginal, perineal and vaginal self‐collected specimens: Implications for vaginal self‐collection
Authors: Belinson Jerome L. , Hu Shangying , Niyazi Mayineur , Pretorius Robert G. , Wang He , Wen Chen , Smith Jennifer S. , Li Jing , Taddeo Frank J. , Burchette Raoul J. , Qiao You‐Lin
Published: December 2009

To determine why a vaginal self‐collection tested for high‐risk human papillomavirus (HR‐HPV) by Hybrid Capture 2® (hc2) has lower sensitivity and specificity for cervical intraepithelial neoplasia Grade 2 or worse (≥CIN 2), we collected 5 specimens (endocervix, upper and lower vagina, perineum, vaginal self‐collection) from 2,625 women. Endocervical and self‐collected specimens had HR‐HPV tests by hc2. All 5 anogenital specimens were tested for 37 HPV genotypes [Linear Array®, (LA)] from 397 women hc2 positive in endocervical or self‐collected specimens and for a randomly selected 71 of 2,228 women hc2 negative on both specimens. Three hundred nintey‐five women who screened positive by hc2 or had abnormal cytology underwent colposcopic evaluation. Of 47 women with ≥CIN 2, hc2 was positive in 97.9% (46/47) of endocervical and 80.9% (38/47), p = 0.008 of self‐collected specimens. Seven of 9 women with ≥CIN 2 and negative self‐collected hc2 tests were positive for HR‐HPV by LA. Of 2,578 women without ≥CIN 2, hc2 was positive in 9.8% (253/2,578) of endocervical and 11.4% (294/2,578), p = 0.001 of self‐collected specimens. Of the 41 more women without ≥CIN 2 that tested hc2 positive on the self‐collected but negative on endocervical specimen, LA tested positive for HR‐HPV in 24, negative for HPV in 11 and negative for HR‐HPV but positive for low‐risk HPV in 6. Lower sensitivity of self‐collected specimens is secondary to lower levels of vaginal HR‐HPV. The principal cause of the lower specificity of self‐collected specimens is HR‐HPV present solely in the vagina, which is not associated with ≥CIN 2.
Muscle atrophy in experimental cancer cachexia: Is the IGF‐1 signaling pathway involved?
Authors: Penna Fabio , Bonetto Andrea , Muscaritoli Maurizio , Costamagna Domiziana , Minero Valerio Giacomo , Bonelli Gabriella , Fanelli Filippo Rossi , Baccino Francesco Maria , Costelli Paola
Published: December 2009

Skeletal muscle wasting, one of the main features of cancer cachexia, is associated with marked protein hypercatabolism, and has suggested to depend also on impaired IGF‐1 signal transduction pathway. To investigate this point, the state of activation of the IGF‐1 system has been evaluated both in rats bearing the AH‐130 hepatoma and in mice transplanted with the C26 colon adenocarcinoma. In the skeletal muscle of tumor hosts, the levels of phosphorylated (active) Akt, one of the most relevant kinases involved in the IGF‐1 signaling pathway, were comparable to controls, or even increased. Accordingly, downstream targets such as GSK3β, p70S6K and FoxO1 were hyperphosphorylated, while the levels of phosphorylated eIF2α were markedly reduced with respect to controls. In the attempt to force the metabolic balance toward anabolism, IGF‐1 was hyperexpressed by gene transfer in the tibialis muscle of the C26 hosts. In healthy animals, IGF‐1 overexpression markedly increased both fiber and muscle size. As a positive control, IGF‐1 was also overexpressed in the muscle of aged mice. In IGF‐1 hyperexpressing muscles the fiber cross‐sectional area definitely increased in both young and aged animals, while, by contrast, loss of muscle mass or reduction of fiber size in mice bearing the C26 tumor were not modified. These results demonstrate that muscle wasting in tumor‐bearing animals is not associated with downregulation of molecules involved in the anabolic response, and appears inconsistent, at least, with reduced activity of the IGF‐1 signaling pathway.
The role of Eph receptors and ephrin ligands in colorectal cancer
Authors: Herath Nirmitha I. , Boyd Andrew W.
Published: December 2009

Eph receptors and their ephrin ligands constitute the largest subfamily of receptor tyrosine kinases and are components of the cell signaling pathways involved during development. Eph and ephrin overexpression have been documented in a variety of human cancers including gastrointestinal malignancies and in particular colorectal malignancies. EphB and ephrin B proteins have been implicated in the homeostasis of the gastrointestinal tract where EphB2‐ and EphB3‐ephrin B signaling regulates cell sorting in the mature epithelium. These proteins are also reported to be upregulated in colon carcinomas. The EphA/ephrin A system has also been implicated in epithelial tissue structure and function. More recently, EphA receptors and their corresponding ligands have been implicated in numerous malignancies. Of these, EphA2 in particular has been intensively investigated and has been proposed as a therapeutic target. An interesting observation emerging from these studies is the role for Ephs and ephrins in critical aspects of cell adhesion, migration and positioning, and a crucial role in tumor progression and metastasis. However, the underlying role of Ephs and ephrins in these processes has generally been studied on individual Eph or ephrin genes. Given the multiplicity of Eph expression on gut epithelial cells, a more global approach is needed to define the precise role of Eph–ephrin interaction in malignant transformation. Here, we will review the recent advances on the role of Eph–ephrin signaling in colorectal malignancies.
Novobiocin decreases SMYD3 expression and inhibits the migration of MDA‐MB‐231 human breast cancer cells
Authors: Luo Xue‐Gang , Zou Jia‐Ning , Wang Shu‐Zhen , Zhang Tong‐Cun , Xi Tao
Published: December 2009

SET and MYND domain‐containing protein 3 (SMYD3) is a histone methyltransferase that plays an important role in transcriptional regulation in human carcinogenesis, and heat‐shock protein HSP90A has been shown to increase the activity of SMYD3. We previously reported that overexpression of SMYD3 stimulated the migration of cells. In this study, we further found that novobiocin, a HSP90 inhibitor, could decrease the expression of SMYD3 and dose dependently inhibit the proliferation and migration of MDA‐MB‐231 human breast cancer cells. As a control, the short hairpin RNA (shRNA) targeting SMYD3 gene also showed similar effects with novobicin. This study is the first to show that novobiocin can inhibit the migration of breast cancer cells and such event may involve the downregulation of SMYD3. These findings might throw light on the development of novel therapeutic approaches to human cancers, and lend further understanding to the potential role of SMYD3 in human carcinogenesis. © 2009 IUBMB IUBMB Life, 62(3): 194–199, 2010
Search for the “ideal analgesic” in pain treatment by engineering the mu‐opioid receptor
Authors: Tao Pao‐Luh , Law Ping‐Yee , Loh Horace H.
Published: December 2009

The myriad of side effects that associate with morphine has been problematic in the clinical use to manage moderate to severe pain. It has been the holy grail of the pharmacologists to develop a compound, or treatment paradigm that could retain the analgesic effect of the drug as eliminating or reducing the side effects, mainly the tolerance and addiction development associates with chronic usage of the drug. In our earlier receptor structure/activities studies, we discovered an unique mutation of a conserved Ser in the fourth transmembrane domain of the opioid receptor that the alkaloid antagonist could activate the receptor. On the basis of this initial finding, we decide to explore the possibility of using virus to deliver the mutant mu‐opioid receptor at the various sites of the nociceptive pathway and induce the antinociceptive responses with the systemic administration of opioid antagonists. In this article, we will summarize the progress of such approach and the probable advantages over the conventional approach of drug development in the treatment of chronic pain. © 2009 IUBMB IUBMB Life, 62(2): 103–111, 2010
Osteoclast‐specific Dicer gene deficiency suppresses osteoclastic bone resorption
Authors: Mizoguchi Fumitaka , Izu Yayoi , Hayata Tadayoshi , Hemmi Hiroaki , Nakashima Kazuhisa , Nakamura Takashi , Kato Shigeaki , Miyasaka Nobuyuki , Ezura Yoichi , Noda Masaki
Published: December 2009

Osteoclasts are unique cells that resorb bone, and are involved in not only bone remodeling but also pathological bone loss such as osteoporosis and rheumatoid arthritis. The regulation of osteoclasts is based on a number of molecules but full details of these molecules have not yet been understood. MicroRNAs are produced by Dicer cleavage an emerging regulatory system for cell and tissue function. Here, we examine the effects of Dicer deficiency in osteoclasts on osteoclastic activity and bone mass in vivo. We specifically knocked out Dicer in osteoclasts by crossing Dicer flox mice with cathepsin K‐Cre knock‐in mice. Dicer deficiency in osteoclasts decreased the number of osteoclasts (N.Oc/BS) and osteoclast surface (Oc.S/BS) in vivo. Intrinsically, Dicer deficiency in osteoclasts suppressed the levels of TRAP positive multinucleated cell development in culture and also reduced NFATc1 and TRAP gene expression. MicroRNA analysis indicated that expression of miR‐155 was suppressed by RANKL treatment in Dicer deficient cells. Dicer deficiency in osteoclasts suppressed osteoblastic activity in vivo including mineral apposition rate (MAR) and bone formation rate (BFR) and also suppressed expression of genes encoding type I collagen, osteocalcin, Runx2, and Efnb2 in vivo. Dicer deficiency in osteoclasts increased the levels of bone mass indicating that the Dicer deficiency‐induced osteoclastic suppression was dominant over Dicer deficiency‐induced osteoblastic suppression. On the other hand, conditional Dicer deletion in osteoblasts by using 2.3 kb type I collagen‐Cre did not affect bone mass. These results indicate that Dicer in osteoclasts controls activity of bone resorption in vivo. J. Cell. Biochem. 109: 866–875, 2010. © 2009 Wiley‐Liss, Inc.
Iejimalides A and B inhibit lysosomal vacuolar H+‐ATPase (V‐ATPase) activity and induce S‐phase arrest and apoptosis in MCF‐7 cells
Authors: McHenry Peter , Wang Wei‐Lin Winnie , Devitt Edward , Kluesner Nicholas , Davisson Vincent Jo , McKee Edward , Schweitzer Dirk , Helquist Paul , Tenniswood Martin
Published: December 2009

Iejimalides are novel macrolides that are cytostatic or cytotoxic against a wide range of cancer cells at low nanomolar concentrations. A recent study by our laboratory characterized the expression of genes and proteins that determine the downstream effects of iejimalide B. However, little is known about the cellular target(s) of iejimalide or downstream signaling that lead to cell‐cycle arrest and/or apoptosis. Iejimalides have been shown to inhibit the activity of vacuolar H+‐ATPase (V‐ATPase) in osteoclasts, but how this inhibition may lead to cell‐cycle arrest and/or apoptosis in epithelial cells is not known. In this study, MCF‐7 breast cancer cells were treated with iejimalide A or B and analyzed for changes in cell‐cycle dynamics, apoptosis, lysosomal pH, cytoplasmic pH, mitochondrial membrane potential, and generation of reactive oxygen species. Both iejimalides A and B sequentially neutralize the pH of lysosomes, induce S‐phase cell‐cycle arrest, and trigger apoptosis in MCF‐7 cells. Apoptosis occurs through a mechanism that involves oxidative stress and mitochondrial depolarization but not cytoplasmic acidification. These data confirm that iejimalides inhibit V‐ATPase activity in the context of epithelial tumor cells, and that this inhibition may lead to a lysosome‐initiated cell death process. J. Cell. Biochem. 109: 634–642, 2010. © 2009 Wiley‐Liss, Inc.
Derivation of murine induced pluripotent stem cells (iPS) and assessment of their differentiation toward osteogenic lineage
Authors: Li Feng , Bronson Sarah , Niyibizi Christopher
Published: December 2009

Induced pluripotent stem cells (iPSCs) have generated hope and excitement because of the potential they possess for generating patient‐specific embryonic‐like stem cells (ESCs). Although many hurdles remain to be solved before the cells can be applied clinically; studies directed toward understanding factors that control differentiation of the cells toward various cell lineages are prerequisites for their future application. In the present study, we generated murine iPSC and assessed their differentiation toward osteogenic lineage. Murine tail tip fibroblasts were reprogrammed into embryonic‐like state by transduction with defined factors (Oct3/4, Sox2, c‐Myc, and klf4) carried in a retroviral vector. The reprogrammed cells expressed ESC markers, gave rise to three germ layers as demonstrated by teratoma formation and immunofluorescence staining. These data confirmed that the reprogrammed cells exhibited ESC‐like state. Treatment of iPSCs‐derived embryoid bodies (EBs) with transforming growth factor beta 1 (TGF‐β1) in the presence of retinoic acid enhanced generation of MSC‐like cells. The MSCs‐like cells expressed putative makers associated with MSCs; the cells deposited calcium in vitro when cultured in osteogenic medium. Interestingly MSCs‐like cells generated from iPSC directed EBs by treatment with retinoic acid and TGF‐β1 deposited more calcium in vitro than cells derived without TGF‐β1 treatment. Taken together, the data demonstrate that iPSC give rise to MSCs‐like state and that the cells have potential to differentiate toward osteoblasts. In addition, brief treatment of iPSC‐derived EBs with TGF‐β1 may be an approach for directing iPSC toward MSC‐like state. J. Cell. Biochem. 109: 643–652, 2010. © 2009 Wiley‐Liss, Inc.
Wnt3‐frizzled 1 chimera as a model to study canonical Wnt signaling
Authors: Bhat Ramesh A. , Stauffer Barbara , Della Pietra Anthony , Bodine Peter V.N.
Published: December 2009

Wnt proteins initiate signaling by binding to seven transmembrane spanning receptors of the frizzled (Fz) family together with the members of the low‐density lipoprotein receptor‐related protein (LRP) 5 and 6. A chimera of human Wnt3 and Fz1 receptor was developed that efficiently activated the TCF‐luciferase reporter. Deletion of the cytoplasmic tail and point mutations in the PDZ binding region in the chimera resulted in the loss of Wnt signaling, suggesting a critical role for the Fz cytoplasmic region in Wnt signaling. The Fz CRD is also critical for Wnt signaling, as a deletion of 29 amino acids in the 2nd cysteine loop resulted in the total loss of TCF‐luciferase activation. DKK‐1 protein blocks upregulation of the TCF‐luciferase reporter by the Wnt3–Fz1 chimera, suggesting involvement of LRP in Wnt3–Fz1 signaling. Expression of a Wnt3–Fz1 chimera in C3H10T1/2 cells resulted in the upregulation of alkaline phosphatase activity and inhibition of adipocyte formation, demonstrating that the Wnt3–Fz1 chimera is a potent activator of differentiation of C3H10T1/2 cells into osteoblasts and an inhibitor of their differentiation into the adipocyte lineage. In summary, the Wnt–Fz chimera approach has the potential to better our understanding of the mechanism of Wnt action and its role, particularly in stem cell differentiation. In addition, this methodology can be utilized to identify inhibitors of either Wnt, Fz or interactors of the canonical pathway, which may have potential therapeutic value in the treatment of cancers and other diseases. J. Cell. Biochem. 109: 876–884, 2010. © 2009 Wiley‐Liss, Inc.
Identification of MicroRNAs controlling human ovarian cell proliferation and apoptosis
Authors: Sirotkin Alexander V. , Lauková Marcela , Ovcharenko Dmitriy , Brenaut Pauline , Mlynček Miloš
Published: December 2009

Previous studies have shown that microRNAs (miRNAs) can control steroidogenesis in cultured granulosa cells. In this study we wanted to determine if miRNAs can also affect proliferation and apoptosis in human ovarian cells. The effect of transfection of cultured primary ovarian granulosa cells with 80 different constructs encoding human pre‐miRNAs on the expression of the proliferation marker, PCNA, and the apoptosis marker, Bax was evaluated by immunocytochemistry. Eleven out of 80 tested miRNA constructs resulted in stimulation, and 53 miRNAs inhibited expression of PCNA. Furthermore, 11 of the 80 miRNAs tested promoted accumulation of Bax, while 46 miRNAs caused a reduction in Bax in human ovarian cells. In addition, two selected antisense constructs that block the corresponding miRNAs mir‐15a and mir‐188 were evaluated for their effects on expression of PCNA. An antisense construct inhibiting mir‐15a (which precursor suppressed PCNA) increased PCNA, whereas an antisense construct for mir‐188 (which precursor did not change PCNA) did not affect PCNA expression. Verification of effects of selected pre‐mir‐10a, mir‐105, and mir‐182 by using other markers of proliferation (cyclin B1) and apoptosis (TdT and caspase 3) confirmed specificity of miRNAs effects on these processes. This is the first direct demonstration of the involvement of miRNAs in controlling both proliferation and apoptosis by ovarian granulose cells, as well as the identification of miRNAs promoting and suppressing these processes utilizing a genome‐wide miRNA screen. J. Cell. Physiol. 223: 49–56, 2010. © 2009 Wiley‐Liss, Inc.
Single‐cell analysis of p16INK4a and p21WAF1 expression suggests distinct mechanisms of senescence in normal human and Li‐Fraumeni Syndrome fibroblasts
Authors: Mirzayans Razmik , Andrais Bonnie , Scott April , Paterson Malcolm C. , Murray David
Published: December 2009

Herein we used single‐cell observation methods to gain insight into the roles of p16INK4A and p21WAF1 (hereafter p16 and p21) in replicative senescence and ionizing radiation‐induced accelerated senescence in human [normal, ataxia telangiectasia (AT) and Li‐Fraumeni syndrome (LFS)] fibroblast strains. Cultures of all strains entered a state of replicative senescence at late passages, as evident from inhibition of growth, acquisition of flattened and enlarged cell morphology, and positive staining for senescence‐associated β‐galactosidase. In addition, proliferating early‐passage cultures of these strains exhibited accelerated senescence in response to ionizing radiation. Immunofluorescence microscopy revealed the heterogeneous expression of p16 in normal and AT fibroblast strains, with the majority of the cells exhibiting undetectable levels of p16 irrespective of in vitro culture age. Importantly, replicative senescence as well as accelerated senescence triggered by ionizing radiation were accompanied by sustained nuclear accumulation of p21, but did not correlate with p16 expression in p53‐proficient (normal and AT) fibroblasts. In p53‐deficient (LFS) fibroblasts, on the other hand, replicative senescence and ionizing radiation‐triggered accelerated senescence strongly correlated with expression of p16 but not of p21. Furthermore, senescence in LFS fibroblasts was associated with genomic instability encompassing polyploidy. Our findings are compatible with a model in which p16 serves as a backup regulator of senescence, triggering this response preferentially in the absence of wild‐type p53 activity. The possibility that one of the tumor‐suppressor functions of p16 may be associated with genomic instability, preventing the emergence of malignant progeny from polyploid giant cells, is also supported by these results. J. Cell. Physiol. 223: 57–67, 2010. © 2009 Wiley‐Liss, Inc.
Multiple Ca2+ signaling pathways regulate intracellular Ca2+ activity in human cardiac fibroblasts
Authors: Chen Jing‐Bo , Tao Rong , Sun Hai‐Ying , Tse Hung‐Fat , Lau Chu‐Pak , Li Gui‐Rong
Published: December 2009

Ca2+ signaling pathways are well studied in cardiac myocytes, but not in cardiac fibroblasts. The aim of the present study is to characterize Ca2+ signaling pathways in cultured human cardiac fibroblasts using confocal scanning microscope and RT‐PCR techniques. It was found that spontaneous intracellular Ca2+ (Ca) oscillations were present in about 29% of human cardiac fibroblasts, and the number of cells with Ca oscillations was increased to 57.3% by application of 3% fetal bovine serum. Ca oscillations were dependent on Ca2+ entry. Ca oscillations were abolished by the store‐operated Ca2+ (SOC) entry channel blocker La3+, the phospholipase C inhibitor U‐73122, and the inositol trisphosphate receptors (IP3Rs) inhibitor 2‐aminoethoxydiphenyl borate, but not by ryanodine. The IP3R agonist thimerosal enhanced Ca oscillations. Inhibition of plasma membrane Ca2+ pump (PMCA) and Na+–Ca2+ exchanger (NCX) also suppressed Ca oscillations. In addition, the frequency of Ca oscillations was reduced by nifedipine, and increased by Bay K8644 in cells with spontaneous Ca2+ oscillations. RT‐PCR revealed that mRNAs for IP3R1‐3, SERCA1‐3, CaV1.2, NCX3, PMCA1,3,4, TRPC1,3,4,6, STIM1, and Orai1‐3, were readily detectable, but not RyRs. Our results demonstrate for the first time that spontaneous Ca oscillations are present in cultured human cardiac fibroblasts and are regulated by multiple Ca2+ pathways, which are not identical to those of the well‐studied contractile cardiomyocytes. This study provides a base for future investigations into how Ca2+ signals regulate biological activity in human cardiac fibroblasts and cardiac remodeling under pathological conditions. J. Cell. Physiol. 223: 68–75, 2010. © 2009 Wiley‐Liss, Inc.
RANKL induces heterogeneous DC‐STAMPlo and DC‐STAMPhi osteoclast precursors of which the DC‐STAMPlo precursors are the master fusogens
Authors: Mensah Kofi A. , Ritchlin Christopher T. , Schwarz Edward M.
Published: December 2009

Osteoclasts (OC) are multinucleated bone resorbing cells that form via RANKL‐induced fusion of heterogeneous mononuclear OC precursors (OCP). Currently, there are no unique surface markers to distinguish these OCP populations, which are diagnostic for erosive and metabolic bone diseases using culture assays. Thus, we investigated expression of DC‐STAMP, a surface receptor required for OCP fusion, during osteoclastogenesis in vitro using a novel monoclonal antibody (1A2). Immunoprecipitation‐Western blot analysis of OCP membrane proteins detected 106 kDa dimeric and 53 kDa monomeric DC‐STAMP in non‐denaturing and denaturing conditions, respectively, with greater sensitivity versus rabbit anti‐sera (KR104). 1A2 also detected 99.9% of undifferentiated monocytes as a single population by flow cytometry with a MFI 100‐fold over background, while KR104 was not useful in this assay. Functionally, 1A2 inhibited OCP fusion in vitro. RANKL stimulation of OCP induced DC‐STAMPlo and DC‐STAMPhi cells, which mature into OC and mononuclear cells respectively as determined by fluorescent microscopy and TRAP assays. Addition of DC‐STAMPhi cells to purified DC‐STAMPlo cultures produced larger, more nucleated OC vs. pure DC‐STAMPlo cultures. RT‐qPCR analysis of these two populations showed that OC markers (Trap and Oc‐stamp) and fusogenic gene expression (Cd9 and Cd47), were significantly increased in DC‐STAMPlo vs. DC‐STAMPhi cells. Collectively, these results demonstrate that DC‐STAMP is expressed on OCP as a dimer, which is efficiently detected by 1A2 via flow cytometry. RANKL induces osteoclastogenesis by stimulating DC‐STAMP internalization in some OCP, and these DC‐STAMPlo cells display the “master fusogen” phenotype. In contrast, DC‐STAMPhi OCP can only act as mononuclear donors. J. Cell. Physiol. 223: 76–83, 2010. © 2009 Wiley‐Liss, Inc.
Bone morphogenetic protein 2 and dexamethasone synergistically increase alkaline phosphatase levels through JAK/STAT signaling in C3H10T1/2 cells
Authors: Mikami Yoshikazu , Asano Masatake , Honda Masaki J. , Takagi Minoru
Published: December 2009

Alkaline phosphatase (ALP) is generally believed to be a faithful marker of osteoblast differentiation, and its expression is induced by bone morphogenetic protein‐2 (BMP‐2) and dexamethasone (Dex). However, the effects of combined administration of BMP‐2 and Dex on ALP transcription have not been extensively examined. In this study, we found that BMP‐2 and Dex synergistically increase ALP levels in mouse C3H10T1/2 pluripotent stem cells. However, switching from one inducer to the other, by adding BMP‐2 or Dex to cell cultures at different times, was no more effective than continuous treatment with either inducer alone. A significant induction of ALP mRNA expression was observed only in cells continuously treated with both inducers. This result suggests that both BMP‐2 and Dex may act in the same pathway or at the same stage of differentiation. A luciferase assay using ALP promoter deletion constructs showed that a region of the promoter containing a putative signal transducer and activator of transcription 3 (STAT3) response element (SRE) responds to treatment with a combination of BMP‐2 and Dex. Furthermore, a ChIP assay indicated that STAT3 bound to the SRE. In addition, a STAT3 siRNA suppressed the synergistic effect of BMP‐2 and Dex on ALP levels. These results indicate that STAT3 may play an important role in regulating ALP expression. To our knowledge, this is the first time that STAT3 has been implicated in the regulation of ALP expression by BMP‐2 and Dex. These findings raise the possibility of developing new strategies for the enhancement of bone formation using a combination of BMPs and Dex. J. Cell. Physiol. 223: 123–133, 2010. © 2009 Wiley‐Liss, Inc.
Laminin‐332–β1 integrin interactions negatively regulate invadopodia
Authors: Liu Shanshan , Yamashita Hironobu , Weidow Brandy , Weaver Alissa M. , Quaranta Vito
Published: December 2009

Adhesion of epithelial cells to basement membranes (BM) occurs through two major structures: actin‐associated focal contacts and keratin‐associated hemidesmosomes, both of which form on laminin‐332 (Ln‐332). In epithelial‐derived cancer cells, additional actin‐linked structures with putative adhesive properties, invadopodia, are frequently present and mediate BM degradation. A recent study proposed that BM invasion requires a proper combination of focal contacts and invadopodia for invading cells to gain traction through degraded BM, and suggested that these structures may compete for common molecular components such as Src kinase. In this study, we tested the role of the Ln‐332 in regulating invadopodia in 804G rat bladder carcinoma cells, a cell line that secretes Ln‐332 and forms all three types of adhesions. Expression of shRNA to Ln‐332 γ2 chain (γ2‐kd) led to increased numbers of invadopodia and enhanced extracellular matrix degradation. Replating γ2‐kd cells on Ln‐332 or collagen‐I fully recovered cell spreading and inhibition of invadopodia. Inhibition of α3 or β1, but not α6 or β4, phenocopied the effect of γ2‐kd, suggesting that α3β1‐mediated focal contacts, rather than α6β4‐mediated hemidesmosome pathways, intersect with invadopodia regulation. γ2‐kd cells exhibited alterations in focal contact‐type structures and in activation of focal adhesion kinase (FAK) and Src kinase. Inhibition of FAK also increased invadopodia number, which was reversible with Src inhibition. These data are consistent with a model whereby actin‐based adhesions can limit the availability of active Src that is capable of invadopodia initiation and identifies Ln‐332‐β1 interactions as a potent upstream regulator that limits cell invasion. J. Cell. Physiol. 223: 134–142, 2010. © 2009 Wiley‐Liss, Inc.