Endosomal recycling and dopamine neurotransmission: Exploring the links between the retromer and Parkinson's disease.

The retromer complex is an evolutionarily conserved protein complex involved in the endosomal recycling of various cargo proteins. It is ubiquitously expressed in all tissue and is found in both invertebrate as well as mammalian nervous systems, where it recycles various synaptic membrane proteins including the dopamine transporter and dopamine D1 receptor, two proteins implicated in dopamine homeostasis and neurotransmission. The involvement of the retromer complex in dopamine neurobiology is further underscored by its links to Parkinson's disease, a neurodegenerative disorder of the dopamine system. In this article, the existing literature linking the retromer complex to synaptic function and dopamine homeostasis is reviewed. Additional possible links are highlighted by exploring the retromer and other Parkinson's disease-associated proteins and possible relationships to synaptic function and dopamine transmission.

Epigenetic events regulating monoallelic gene expression.

In mammals, generally it is assumed that the genes inherited from each parent are expressed to similar levels. However, it is now apparent that in non-sex chromosomes, 6-10% of genes are selected for monoallelic expression. Monoallelic expression or allelic exclusion is established either in an imprinted (parent-of-origin) or a stochastic manner. The stochastic model explains random selection while the imprinted model describes parent-of-origin specific selection of alleles for expression. Allelic exclusion occurs during X chromosome inactivation, parent-of-origin expression of imprinted genes and stochastic monoallelic expression of cell surface molecules, clustered protocadherin (PCDH) genes. Mis-regulation or loss of allelic exclusion contributes to developmental diseases. Epigenetic mechanisms are fundamental players that determine this type of expression despite a homogenous genetic background. DNA methylation and histone modifications are two mediators of the epigenetic phenomena. The majority of DNA methylation is found on cytosines of the CpG dinucleotide in mammals. Several covalent modifications of histones change the electrostatic forces between DNA and histones modifying gene expression. Long-range chromatin interactions organize chromatin into transcriptionally permissive and prohibitive regions leading to simultaneous regulation of gene expression and repression. Non-coding RNAs (ncRNAs) are also players in regulating gene expression. Together, these epigenetic mechanisms fine-tune gene expression levels essential for normal development and survival. In this review, first we discuss what is known about monoallelic gene expression. Then, we focus on the molecular mechanisms that regulate expression of three monoallelically expressed gene classes: the X-linked genes, selected imprinted genes and PCDH genes.

An integrated microfluidic chip for immunomagnetic detection and isolation of rare prostate cancer cells from blood.

The quantitative and qualitative analysis of circulating tumor cells (CTCs) has the potential to improve the clinical management of several cancers, including prostate cancer. As such, there is much interest in the isolation of CTCs from the peripheral blood of cancer patients. We report the design, fabrication, and proof-of-principle testing of an integrated permalloy-based microfluidic chip for immunomagnetic isolation of blood-borne prostate cancer cells using an antibody targeting prostate surface membrane antigen (PSMA). The preliminary results using spiked blood samples indicate that the proposed device is consistently capable of isolating prostate cancer cells with high sensitivity (up to 98 %) at clinically relevant low concentrations (down to 20 cells/mL) and an acceptable throughput (100 µL/min).

Induction of Cytotoxicity in Pyridine Analogues of the Anti-metastatic Ru(III) Complex NAMI-A by Ferrocene Functionalization.

A series of novel ferrocene (Fc) functionalized Ru(III) complexes was synthesized and characterized. These compounds are derivatives of the anti-metastatic Ru(III) complex imidazolium [trans-RuCl4(1H-imidazole) (DMSO-S)] (NAMI-A) and are derived from its pyridine analogue (NAMI-Pyr), with direct coupling of Fc to pyridine at the 4 or 3 positions, or at the 4 position via a two-carbon linker, which is either unsaturated (vinyl) or saturated (ethyl). Electron paramagnetic resonance (EPR) and UV-vis spectroscopic studies of the ligand exchange processes of the compounds in phosphate buffered saline (PBS) report similar solution behavior to NAMI-Pyr. However, the complex with Fc substitution at the 3 position of the coordinated pyridine shows greater solution stability, through resistance to the formation of oligomeric species. Further EPR studies of the complexes with human serum albumin (hsA) indicate that the Fc groups enhance noncoordinate interactions with the protein and help to inhibit the formation of protein-coordinated species, suggesting the potential for enhanced bioavailability. Cyclic voltammetry measurements demonstrate that the Fc groups modestly reduce the reduction potential of the Ru(III) center as compared to NAMI-Pyr, while the reduction potentials of the Fc moieties of the four compounds vary by 217 mV, with the longer linkers giving significantly lower values of E1/2. EPR spectra of the compounds with 2-carbon linkers show the formation of a high-spin Fe(III) species (S = 5/2) in PBS with a distinctive signal at g = 4.3, demonstrating oxidation of the Fe(II) ferrocene center and likely reflecting degradation products. Density functional theory calculations and paramagnetic (1)H NMR describe delocalization of spin density onto the ligands and indicate that the vinyl linker could be a potential pathway for electron transfer between the Ru and Fe centers. In the case of the ethyl linker, electron transfer is suggested to occur via an indirect mechanism enabled by the greater flexibility of the ligand. In vitro assays with the SW480 cell line reveal cytotoxicity induced by the ruthenium ferrocenylpyridine complexes that is at least an order of magnitude higher than the unfunctionalized complex, NAMI-Pyr. Furthermore, migration studies with LNCaP cells reveal that Fc functionalization does not reduce the ability of the compounds to inhibit cell motility. Overall, these studies demonstrate that NAMI-A-type compounds can be functionalized with redox-active ligands to produce both cytotoxic and anti-metastatic activity.

The Flame-Retardant Tris(1,3-dichloro-2-propyl) Phosphate Represses Androgen Signaling in Human Prostate Cancer Cell Lines.

The effects of six organophosphate flame retardants (OPFRs) tris(2-butoxyethyl) phosphate, tris(2-chloroethyl) phosphate, tris(1-chloro-2-propyl) phosphate, tris(methylphenyl) phosphate, tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), and triethyl phosphate on the activities of androgen receptor (AR), estrogen receptor (ER), and aryl hydrocarbon receptor (AhR) were assessed in human prostate and endometrial cancer cells. OPFRs had no effect on ER or AhR target gene activation in ECC-1 cells. The effect of TDCIPP on mRNA and protein accumulation of AR target genes was examined further. AR-inducible gene and protein expression were significantly altered by TDCIPP exposure and repressed PSA levels in conditioned media of prostate cancer cells. We demonstrated that TDCIPP has no affinity for the AR ligand binding domain (AR-LBD) and exerts its antiandrogenic effects in a noncompetitive fashion. Thus, the clinical relevance of TDCIPP exposure on prostate cancer detection and progression to a therapeutically refractile state ought to be investigated further.

The Effects of the Organic Flame-Retardant 1,2-Dibromo-4-(1,2-dibromoethyl) Cyclohexane (TBECH) on Androgen Signaling in Human Prostate Cancer Cell Lines.

The effects of the organic flame retardant 1,2-Dibromo-4-(1,2-dibromoethyl) cyclohexane (TBECH) on androgen receptor target gene expression were examined in the human LNCaP prostate cancer cell line. While γ-/δ-TBECH alone led to a significant increase in prostate-specific antigen (PSA) mRNA accumulation, both the α-/-TBECH and γ-/δ-TBECH mixtures repressed androgen-inducible PSA mRNA and protein accumulation in human LNCaP cells. Thus, we hypothesize that isomeric mixtures of TBECH may act as partial agonists of the androgen receptor.

Estrogen receptor expression is required for low-dose resveratrol-mediated repression of aryl hydrocarbon receptor activity.

The putative cardioprotective and chemopreventive properties of the red wine phenolic resveratrol (RES) have made it the subject of a growing body of clinical and basic research. We have begun investigations focusing on the effects of RES on the activity of the aryl hydrocarbon receptor (AHR) complex. Our evidence suggests that RES is a potent repressor of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible gene transcription in estrogen receptor (ER)-positive human breast, lung, and colon cancer cell lines. RES activates the transcription of the ER target genes to the same degree as estradiol (E(2)) in human MCF-7 breast cancer cells. Unlike E(2), which can only diminish TCDD-inducible CYP1A1 gene transcription by approximately 50%, RES can completely abrogate this response. Furthermore, 50% repression of TCDD-inducible transcription can be achieved with 100 nM RES, approximately 2.5 orders of magnitude lower than concentrations required for maximal inhibition, suggesting that multiple mechanisms are responsible for this effect. RES (100 nM) does not prevent ligand binding of a TCDD analog, nor does it prevent AHR from binding to its response element in the 5'-regulatory region of the CYP1A1 gene. Small inhibitory RNAs directed to ERα have demonstrated that RES-mediated repression of CYP1A1 depends on ERα. Whereas CYP1A1 protein levels in MCF-7 cells are refractory to the low-dose transcriptional effects of RES, a concomitant decrease in CYP1A1 protein levels is observed in Caco-2 cells. These results highlight a low-dose RES effect that could occur at nutritionally relevant exposures and are distinct from the high-dose effects often characterized.

Development of a microfluidic platform for size-based hydrodynamic enrichment and PSMA-targeted immunomagnetic isolation of circulating tumour cells in prostate cancer.

Efforts to further improve the clinical management of prostate cancer (PCa) are hindered by delays in diagnosis of tumours and treatment deficiencies, as well as inaccurate prognoses that lead to unnecessary or inefficient treatments. The quantitative and qualitative analysis of circulating tumour cells (CTCs) may address these issues and could facilitate the selection of effective treatment courses and the discovery of new therapeutic targets. Therefore, there is much interest in isolation of elusive CTCs from blood. We introduce a microfluidic platform composed of a multiorifice flow fractionation (MOFF) filter cascaded to an integrated microfluidic magnetic (IMM) chip. The MOFF filter is primarily employed to enrich immunomagnetically labeled blood samples by size-based hydrodynamic removal of free magnetic beads that must originally be added to samples at disproportionately high concentrations to ensure the efficient immunomagnetic labeling of target cancer cells. The IMM chip is then utilized to capture prostate-specific membrane antigen-immunomagnetically labeled cancer cells from enriched samples. Our preclinical studies showed that the proposed method can selectively capture up to 75% of blood-borne PCa cells at clinically-relevant low concentrations (as low as 5 cells/ml), with the IMM chip showing up to 100% magnetic capture capability.

The microfluidic capture of single breast cancer cells for multi-drug resistance assays.

Utilizing the microfluidic single-cell technique enables us to study the inhibition of multidrug resistance due to drug efflux on a single triple-negative breast cancer cell. This method examines drug efflux inhibition on a single cell in a microfluidic chip using a conventional optical detection system constructed from an inverted microscope and a microphotometer. More importantly, the integration of single-cell selection, dye and drug loading, and fluorescence measurement for intracellular drug accumulation is all conducted on a single microfluidic chip. By using a microfluidic chip and the adherent nature of the cancer cell lines, a single breast cancer cell could be selected and retained near the cell retention structure in the chip. This enabled us to detect dye accumulation in the MDR breast cells in the presence of cyclosporine A (CsA). CsA and rhodamine 123 (Rh123) were used as the P-glycoprotein (P-gp) inhibitor and fluorescent dye, respectively. Furthermore, Paclitaxel, a commonly known chemotherapeutic used in breast cancer patients, was administered in the presence of both reagents. During the entirety of the experiment fluorescence measurement was used to monitor the fluctuating levels of intracellular Rh123 levels, and an optical imaging system was used to monitor the shape and size of the cell. The results showed that the Rh123 fluorescence signal in a single-cell increased dramatically over its same-cell control due to the competitive inhibition of paclitaxel and the non-competitive inhibition subjected by CsA.

Expression of human inducible nitric oxide synthase in response to cytokines is regulated by hypoxia-inducible factor-1.

The production of nitric oxide (NO) by inducible NO synthase (iNOS) and the regulation of gene expression by hypoxia-inducible factors (HIFs) are important for many aspects of human cell biology. However, little is known about whether iNOS expression is controlled by HIFs in human cells. Stimulation of A549 human lung epithelial cells with cytokines (TNF, IL-1 and IFNγ) increased the nuclear accumulation of HIF-1 in normoxic conditions. Activation of HIF-1 by hypoxia or CoCl2 was not sufficient to induce iNOS expression. However, pharmacological inhibition of HIF-1 reduced the induction of iNOS expression in A549 cells and primary human astrocytes. Moreover, elimination of HIF-1α expression and activity by CRISPR/Cas9 gene editing significantly reduced the induction of human iNOS gene promoter, mRNA and protein expression by cytokine stimulation. Three putative hypoxia response elements (HRE) are present within the human iNOS gene promoter and elimination of an HRE at -4981 bp reduced the induction of human iNOS promoter activity in response to cytokine stimulation. These findings establish an important role for HIF-1α in the induction of human iNOS gene expression in response to cytokine stimulation.

The aryl hydrocarbon receptor complex and the control of gene expression.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that controls the expression of a diverse set of genes. The toxicity of the potent AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin is almost exclusively mediated through this receptor. However, the key alterations in gene expression that mediate toxicity are poorly understood. It has been established through characterization of AhR-null mice that the AhR has a required physiological function, yet how endogenous mediators regulate this orphan receptor remains to be established. A picture as to how the AhR/ARNT heterodimer actually mediates gene transcription is starting to emerge. The AhR/ARNT complex can alter transcription both by binding to its cognate response element and through tethering to other transcription factors. In addition, many of the coregulatory proteins necessary for AhR-mediated transcription have been identified. Cross talk between the estrogen receptor and the AhR at the promoter of target genes appears to be an important mode of regulation. Inflammatory signaling pathways and the AhR also appear to be another important site of cross talk at the level of transcription. A major focus of this review is to highlight experimental efforts to characterize nonclassical mechanisms of AhR-mediated modulation of gene transcription.

ChIP-re-ChIP: Co-occupancy Analysis by Sequential Chromatin Immunoprecipitation.

Chromatin immunoprecipitation (ChIP) exploits the specific interactions between DNA and DNA-associated proteins. It can be used to examine a wide range of experimental parameters. A number of proteins bound at the same genomic location can identify a multi-protein chromatin complex where several proteins work together to regulate gene transcription or chromatin configuration. In many instances, this can be achieved using sequential ChIP; or simply, ChIP-re-ChIP. Whether it is for the examination of specific transcriptional or epigenetic regulators, or for the identification of cistromes, the ability to perform a sequential ChIP adds a higher level of power and definition to these analyses. In this chapter, we describe a simple and reliable method for the sequential ChIP assay.

Recruitment of the NCoA/SRC-1/p160 family of transcriptional coactivators by the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator complex.

The aryl hydrocarbon receptor complex heterodimeric transcription factor, comprising the basic helix-loop-helix-Per-ARNT-Sim (bHLH-PAS) domain aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) proteins, mediates the toxic effects of TCDD (2,3,7,8 tetrachlorodibenzo-p-dioxin). The molecular events underlying TCDD-inducible gene activation, beyond the activation of the AHRC, are poorly understood. The SRC-1/NCoA-1, NCoA-2/GRIP-1/TIF-2, and p/CIP/AIB/ACTR proteins have been shown to act as mediators of transcriptional activation. In this report, we demonstrate that SRC-1, NCoA-2, and p/CIP are capable of independently enhancing TCDD-dependent induction of a luciferase reporter gene by the AHR/ARNT dimer. Furthermore, injection of anti-SRC-1 or anti-p/CIP immunoglobulin G into mammalian cells abolishes the transcriptional activity of a TCDD-dependent reporter gene. We demonstrate by coimmunoprecipitation and by a reporter gene assay that SRC-1 and NCoA-2 but not p/CIP are capable of interacting with ARNT in vivo after transient transfection into mammalian cells, while AHR is capable of interacting with all three coactivators. We confirm the interactions of ARNT and AHR with SRC-1 with immunocytochemical techniques. Furthermore, SRC-1, NCoA-2, and p/CIP all associate with the CYP1A1 enhancer region in a TCDD-dependent fashion, as demonstrated by chromatin immunoprecipitation assays. We demonstrate by yeast two-hybrid, glutathione S-transferase pulldown, and mammalian reporter gene assays that ARNT requires its helix 2 domain but not its transactivation domain to interact with SRC-1. This indicates a novel mechanism of action for SRC-1. SRC-1 does not require its bHLH-PAS domain to interact with ARNT or AHR, but utilizes distinct domains proximal to its p300/CBP interaction domain. Taken together, these data support a role for the SRC family of transcriptional coactivators in TCDD-dependent gene regulation.

Glioma: Tryptophan Catabolite and Melatoninergic Pathways Link microRNA, 14-3- 3, Chromosome 4q35, Epigenetic Processes and other Glioma Biochemical Changes.

Primary glioma, as well as secondary metastases, provide significant treatment challenges. An understanding of the biological underpinnings of glioma is likely to provide new pharmaceutical targets that will improve patient survival. Here, we look at the role that the kynurenine pathways and associated tyrptophan catabolites (TRYCATs) play in glioma, linking this to changes in oxidative and nitrosative stress (O&NS), immuneinflammatory activity, the aryl hydrocarbon receptor (AhR), and the melatoninergic pathways. It is suggested that the interactions of O&NS and the immune-inflammatory processes in glioma contribute to the induction of the TRYCATs via the kynurenine activation of the AhR, leading to increased indoleamine 2,3-dioxygenase, which deprives tryptophan for the necessary serotonin that is required as a precursor for the melatoninergic pathways. A diverse array of data pertaining to glioma can be linked to these pathways, including changes in miRNAs, epigenetic processes, estrogen receptors, 14-3-3, chromosome 4q35, neurotrophins, tristetraprolin and the N-acetylserotonin (NAS)/melatonin ratio. As many of these factors directly or indirectly act on the melatoninergic pathways, including variations in the NAS/melatonin ratio, it is suggested that the melatoninergic pathways may act as a hub that co-ordinate the multitude of changes occurring in glioma. Consequently, the melatoninergic pathways may be a significant pharmaceutical target for the treatment of this still very poorly managed condition.

The retinoblastoma protein regulates hypoxia-inducible genetic programs, tumor cell invasiveness and neuroendocrine differentiation in prostate cancer cells.

Loss of tumor suppressor proteins, such as the retinoblastoma protein (Rb), results in tumor progression and metastasis. Metastasis is facilitated by low oxygen availability within the tumor that is detected by hypoxia inducible factors (HIFs). The HIF1 complex, HIF1α and dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT), is the master regulator of the hypoxic response. Previously, we demonstrated that Rb represses the transcriptional response to hypoxia by virtue of its association with HIF1. In this report, we further characterized the role Rb plays in mediating hypoxia-regulated genetic programs by stably ablating Rb expression with retrovirally-introduced short hairpin RNA in LNCaP and 22Rv1 human prostate cancer cells. DNA microarray analysis revealed that loss of Rb in conjunction with hypoxia leads to aberrant expression of hypoxia-regulated genetic programs that increase cell invasion and promote neuroendocrine differentiation. For the first time, we have established a direct link between hypoxic tumor environments, Rb inactivation and progression to late stage metastatic neuroendocrine prostate cancer. Understanding the molecular pathways responsible for progression of benign prostate tumors to metastasized and lethal forms will aid in the development of more effective prostate cancer therapies.

Probing mechanical properties of Jurkat cells under the effect of ART using oscillating optical tweezers.

Acute lymphoid leukemia is a common type of blood cancer and chemotherapy is the initial treatment of choice. Quantifying the effect of a chemotherapeutic drug at the cellular level plays an important role in the process of the treatment. In this study, an oscillating optical tweezer was employed to characterize the frequency-dependent mechanical properties of Jurkat cells exposed to the chemotherapeutic agent, artesunate (ART). A motion equation for a bead bound to a cell was applied to describe the mechanical characteristics of the cell cytoskeleton. By comparing between the modeling results and experimental results from the optical tweezer, the stiffness and viscosity of the Jurkat cells before and after the ART treatment were obtained. The results demonstrate a weak power-law dependency of cell stiffness with frequency. Furthermore, the stiffness and viscosity were increased after the treatment. Therefore, the cytoskeleton cell stiffness as the well as power-law coefficient can provide a useful insight into the chemo-mechanical relationship of drug treated cancer cells and may serve as another tool for evaluating therapeutic performance quantitatively.

p,p'-Dichlorodiphenyltrichloroethane (p,p'-DDT) and p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) repress prostate specific antigen levels in human prostate cancer cell lines.

Despite stringent restrictions on their use by many countries since the 1970s, the endocrine disrupting chemicals, DDT and DDE are still ubiquitous in the environment. However, little attention has been directed to p,p'-DDT and the anti-androgen, p,p'-DDE on androgen receptor (AR) target gene transcription in human cells. Inhibitors of androgenic activity may have a deleterious clinical outcome in prostate cancer screens and progression, therefore we determined whether environmentally relevant concentrations of p,p'-DDT and p,p'-DDE negatively impact AR-regulated expression of prostate-specific antigen (PSA), and other AR target genes in human LNCaP and VCaP prostate cancer cells. Quantitative real-time PCR and immuno-blotting techniques were used to measure intracellular PSA, PSMA and AR mRNA and protein levels. We have shown for the first time that p,p'-DDT and p,p'-DDE repressed R1881-inducible PSA mRNA and protein levels in a dose-dependent manner. Additionally, we used the fully automated COBAS PSA detection system to determine that extracellular PSA levels were also significantly repressed. These chemicals achieve this by blocking the recruitment of AR to the PSA promoter region at 10 µM, as demonstrated by the chromatin immunoprecipitation (ChIP) in LNCaP cells. Both p,p'-DDT and p,p'-DDE repressed R1881-inducible AR protein accumulation at 10 µM. Thus, we conclude that men who have been exposed to either DDT or DDE may produce a false-negative PSA test when screening for prostate cancer, resulting in an inaccurate clinical diagnosis. More importantly, prolonged exposure to these anti-androgens may mimic androgen ablation therapy in individuals with prostate cancer, thus exacerbating the condition by inadvertently forcing adaptation to this stress early in the disease.

Mechanical characterization of ART-treated Jurkat cells using optical tweezers.

Acute lymphoid leukemia is a common type of blood cancer and chemotherapy is the initial treatment of choice. Quantifying the effectiveness of a chemotherapeutic drug at the cellular level plays an important role in the process of the treatment. In this study, an optical tweezer was employed to characterize the mechanical properties of Jurkat cells exposed to artesunate (ART) as a chemotherapy. A mathematical model was developed to describe the mechanical characteristics of the cell membrane and its features. By comparing the modeling results against experimental results from the optical tweezer, the elastic modulus of the Jurkat cells before and after ART treatment was calculated. The results demonstrate an increase in the cell stiffness after treatment. Therefore, the elastic modulus of a cell membrane may be a useful biomarker to quantify the effectiveness of a chemotherapeutic agent.

Epigenetic characterization of the growth hormone gene identifies SmcHD1 as a regulator of autosomal gene clusters.

Regulatory elements for the mouse growth hormone (GH) gene are located distally in a putative locus control region (LCR) in addition to key elements in the promoter proximal region. The role of promoter DNA methylation for GH gene regulation is not well understood. Pit-1 is a POU transcription factor required for normal pituitary development and obligatory for GH gene expression. In mammals, Pit-1 mutations eliminate GH production resulting in a dwarf phenotype. In this study, dwarf mice illustrated that Pit-1 function was obligatory for GH promoter hypomethylation. By monitoring promoter methylation levels during developmental GH expression we found that the GH promoter became hypomethylated coincident with gene expression. We identified a promoter differentially methylated region (DMR) that was used to characterize a methylation-dependent DNA binding activity. Upon DNA affinity purification using the DMR and nuclear extracts, we identified structural maintenance of chromosomes hinge domain containing -1 (SmcHD1). To better understand the role of SmcHD1 in genome-wide gene expression, we performed microarray analysis and compared changes in gene expression upon reduced levels of SmcHD1 in human cells. Knock-down of SmcHD1 in human embryonic kidney (HEK293) cells revealed a disproportionate number of up-regulated genes were located on the X-chromosome, but also suggested regulation of genes on non-sex chromosomes. Among those, we identified several genes located in the protocadherin ß cluster. In addition, we found that imprinted genes in the H19/Igf2 cluster associated with Beckwith-Wiedemann and Silver-Russell syndromes (BWS & SRS) were dysregulated. For the first time using human cells, we showed that SmcHD1 is an important regulator of imprinted and clustered genes.

A TRIP230-retinoblastoma protein complex regulates hypoxia-inducible factor-1α-mediated transcription and cancer cell invasion.

Localized hypoxia in solid tumors activates transcriptional programs that promote the metastatic transformation of cells. Like hypoxia-inducible hyper-vascularization, loss of the retinoblastoma protein (Rb) is a trait common to advanced stages of tumor progression in many metastatic cancers. However, no link between the role of Rb and hypoxia-driven metastatic processes has been established. We demonstrated that Rb is a key mediator of the hypoxic response mediated by HIF1α/ß, the master regulator of the hypoxia response, and its essential co-activator, the thyroid hormone receptor/retinoblastoma-interacting protein (TRIP230). Furthermore, loss of Rb unmasks the full co-activation potential of TRIP230. Using small inhibitory RNA approaches in vivo, we established that Rb attenuates the normal physiological response to hypoxia by HIF1α. Notably, loss of Rb results in hypoxia-dependent biochemical changes that promote acquisition of an invasive phenotype in MCF7 breast cancer cells. In addition, Rb is present in HIF1α-ARNT/HIF1ß transcriptional complexes associated with TRIP230 as determined by co-immuno-precipitation, GST-pull-down and ChIP assays. These results demonstrate that Rb is a negative modulator of hypoxia-regulated transcription by virtue of its direct effects on the HIF1 complex. This work represents the first link between the functional ablation of Rb in tumor cells and HIF1α-dependent transcriptional activation and invasion.