In vitro activities of novel 4-HPR derivatives on a panel of rhabdoid and other tumor cell lines

<p>Abstract</p> <p>Background</p> <p>Rhabdoid tumors (RTs) are aggressive pediatric malignancies with poor prognosis. N-(4-hydroxy phenyl) retinamide (4-HPR or fenretinide) is a potential chemotherapeutic for RTs with activity correlated to its ability to down-modulate Cyclin D1. Previously, we synthesized novel halogen-substituted and peptidomimetic-derivatives of 4-HPR that retained activity in MON RT cells. Here we analyzed the effect of 4-HPR in inhibiting the growth of several RT, glioma, and breast cancer cell lines and tested their effect on cell cycle, apoptosis and Cyclin D1 expression.</p> <p>Methods</p> <p>Effect of compounds on RT cell cycle profiles, and cell death were assessed by MTS cell survival assays and FACS analysis. The effects of treatment on Cyclin D1 expression were determined by immunoblotting. The efficacy of these compounds on glioma and breast cancer cell lines was also determined using MTS assays.</p> <p>Results</p> <p>Low micromolar concentrations of 4-HPR derivatives inhibited cell survival of all RT cells tested. The 4-HPR derivatives altered RT cell cycle profiles and induced high levels of cell death that was correlated with their potency. ATRA exhibited high IC₅₀values in all cell lines tested and did not cause cell death. In MON RT cells, the iodo-substituted compounds were more active than 4-HPR in inducing cell cycle arrest and apoptosis. Additionally, the activity of the compounds correlated with their ability to down-modulate Cyclin D1: while active compounds reduced Cyclin D1 levels, inactive ATRA did not. In glioma and breast cancer cell lines, 4-HPR and 4-HPR derivatives showed variable efficacy.</p> <p>Conclusions</p> <p>Here we demonstrate, for the first time, that the inhibitory activities of novel halogen-substituted and peptidomimetic derivatives of 4-HPR are correlated to their ability to induce cell death and down-modulate Cyclin D1. These 4-HPR derivatives showed varied potencies in breast cancer and glioma cell lines. These data indicate that further studies are warranted on these derivatives of 4-HPR due to their low IC₅₀s in RT cells. These derivatives are of general interest, as conjugation of halogen radioisotopes such as ¹⁸F, ¹²⁴I, or ¹³¹I to 4-HPR will allow us to combine chemotherapy and radiotherapy with a single drug, and to perform PET/SPECT imaging studies in the future.</p

HIV-1 replication in cell lines harboring INI1/hSNF5 mutations

BACKGROUND: INI1/hSNF5 is a cellular protein that directly interacts with HIV-1 integrase (IN). It is specifically incorporated into HIV-1 virions. A dominant negative mutant derived from INI1 inhibits HIV-1 replication. Recent studies indicate that INI1 is associated with pre-integration and reverse transcription complexes that are formed upon viral entry into the target cells. INI1 also is a tumor suppressor, biallelically deleted/mutated in malignant rhabdoid tumors. We have utilized cell lines derived from the rhabdoid tumors, MON and STA-WT1, that harbor either null or truncating mutations of INI1 respectively, to assess the effect of INI1 on HIV-1 replication. RESULTS: We found that while HIV-1 virions produced in 293T cells efficiently transduced MON and STA-WT1 cells, HIV-1 particle production was severely reduced in both of these cells. Reintroduction of INI1 into MON and STA-WT1 significantly enhanced the particle production in both cell lines. HIV-1 particles produced in MON cells were reduced for infectivity, while those produced in STA-WT1 were not. Further analysis indicated the presence of INI1 in those virions produced from STA-WT1 but not from those produced from MON cells. HIV-1 produced in MON cells were defective for synthesis of early and late reverse transcription products in the target cells. Furthermore, virions produced in MON cells were defective for exogenous reverse transcriptase activity carried out using exogenous template, primer and substrate. CONCLUSION: Our results suggest that INI1-deficient cells exhibit reduced particle production that can be partly enhanced by re-introduction of INI1. Infectivity of HIV-1 produced in some but not all INI1 defective cells, is affected and this defect may correlate to the lack of INI1 and/or some other proteins in these virions. The block in early events of virion produced from MON cells appears to be at the stage of reverse transcription. These studies suggest that presence of INI1 or some other host factor in virions and reverse transcription complexes may be important for early events of HIV-1 replication

Regulation of rat Liver Glucokinase Gene Expression by Sterol Regulatory Element Binding Protein-1a and Forkhead box classO1 Transcription factors

Die Glucokinase (GK), auch Hexokinase-IV, katalysiert die Phosphorylierung von Glucose zu Glucose-6-phosphat. Im Gengensatz zu anderen Hexokinasen hat die GK eine geringe Affinität für Glucose, wird durch das Reaktionsprodukt nicht gehemmt und weist eine sigmoidale Kinetik auf, obwohl sie als Monomer vorliegt. Defekte im GK-Gen führen zum maturity-onset diabetes of the young type-2 (Insulin-unabhängig [MODY-2]). So spielt die GK eine wichtige Rolle bei der Aufrechterhaltung der Glucose-Homeostase. Die GK wird vorwiegend in Leber-Hepatozyten, β-Zellen des Pankreas und in einigen neuroendokrinen Zellen des Gastrolintestinaltrakts und des Gehirns exprimiert. In Hepatozyten sind hauptsächlich Insulin und Glucagon für die Regulation der GK-Expression verantwortlich. Hierbei agiert Insulin vorwiegend über den PI3K/PKB-signalweg und moduliert die Aktivität verschiedener Transkriptionsfaktoren, wie z.B. dem Sterol regulatory element binding protein-1 (SREBP-1) und den FoxO/forkhead Transkriptionsfaktoren. Neuere Veröffentlichungen deuten darauf hin, dass sowohl SREBP-1 als auch FoxO1 in antagonistischer Weise die GK-Expression und somit den Glucose-/Lipid-Metabolismus in der Leber beeinflussen. Die genauen Details der SREBP-1 und FoxO1-regulierten GK-Genexpression sind allerdings noch nicht bekannt. Das Ziel dieser Arbeit war daher die Untersuchung der Insulin-abhängigen SREBP-1 und FoxO1- vermittelten Expression des GK-Gens in primären Rattenhepatozyten und in den Hepatomazellen HepG2. Die Stimulation von primaren Rattenhepatozyten mit Insulin induzierte die Expression von GK und SREBP-1. Auch konnte SREBP-1 durch eine Behandlung mit dem LXR-Agonisten TO-901317 induziert werden, was wiederum zu einer Induktion der GK-mRNA Spiegel führte. Computer-Analysen des Leber-spezifischen GK-Promotors zeigten 3 putative SREBP-1 Bindungsstellen (SREs) auf. Transfektionsversuche mit Luciferasegen-Konstrukten, welche durch seriell deletierte GK-Promotoren reguliert werden, deuteten darauf hin, dass eine Sequenz, bekannt als Footprint-B-Seite, in die SREBP-1-abhängige Regulation involviert ist. Weitere Analysen der Footprint-B-Seite, welche in Abschnitt 1 und Abschnitt 2 unterteilt werden kann, zeigten, dass vor allem Abschnitt 2 und weniger Abschnitt 1 für den SREBP-1 Effekt notwendig ist. Des Weiteren konnten durch Mutationsanalysen des GK-Promotors zwei andere Sequenzen, SRE2 und SRE3, identifiziert werden. Transfektionsversuche in primären Hepatozyten und in HepG2-Zellen implizieren, dass diese Elemente Zell-spezifisch verwendet werden. Während in primären Hepatozyten sowohl SRE2 als auch SRE3 für die SREBP-1 vermittelte GK-Promotoraktivität wichtig sind, trägt in HepG2-Zellen nur SRE2 zum SREBP-1-Effekt bei. Zudem ging die SREBP-1-vermittelte Aktivierung des GK-Promotors durch Mutation des HNF-4-Bindungselements verloren, was darauf hindeutet, dass für die vollständige Induktion der GK-Genexpression durch SREBP-1 eine Interaktion dieser Transkriptionsfaktoren notwendig ist. Insulin hat einen dynamischen Effekt auf die FoxO-Transkriptionsfaktoren, welche durch eine PKB-abhängige Phosphorylierung in eine inaktive Form überführt werden und so aus dem Kern ausgeschlossen werden. Hepatozyten, welche mit FoxO1-Expressionsvektoren transfiziert wurden, regulierten ihre GK-mRNA und GK-Promotoraktivität herab. Diese Repression ging durch Stimulation der Hepatozyten mit Insulin verloren. Bekamen Ratten für 48h kein Futter, so war das GK-Protein kaum detektierbar, allerdings war der FoxO1 Proteinspiegel erhöht. Es ist bekannt, dass die transkriptionelle Aktivität der FoxO-Proteine neben Insulin auch durch NAD+ abhängige SIRT1 Deacetylierung reguliert wird. Resveratrol reguliert die mRNA- und Proteinspiegel der GK herab und kehrt den induzierenden Effekt von Insulin um. Ähnliche Resultate wurden für die GK-Enzymaktivität beobachtet. Computer-Analysen des GK-Promotors prognostizierten zwei FoxO1-Bindungselemente (FBEa und FBEb). Überexpression von FoxO1 vermindert die GK-Promotoraktivität in primaren Hepatozyten und in HepG2-Zellen. Mutationen im FoxO1-Bindungselement FBEb beseitigen die FoxO1 vermittelte Repression des GK-Promotors. Des Weiteren führt die Behandlung von Hepatozyten mit dem SIRT1-Aktivator Resveratrol zur Deacetylierung und Aktivierung von FoxO1. Interessanter Weise ging der FoxO1-Effekt auch verloren, wenn die HNF4-Bindungsstelle mutiert war. Dies suggerierte, das FoxO1 mit HNF-4 interagiert und dessen Aktivität reguliert. Insgesamt zeigt die Studie, dass SREBP-1 die GK-Genexpression über die Footprint-B2-Seite, SRE2 und über Interaktion mit HNF-4 aktivieren kann. Obwohl die Interaktion mit HNF-4 auch für die FoxO1-abhängige Regulation des GK- Promotor wichtig ist, konnten zwei zusätzliche Bindungsstellen identifiziert werden. Zum ersten Mal konnte gezeigt weden, dass die FoxO1-Aktivität durch Resveratrol und SIRT1 reguliert wird und dass die Resveratrol-vermittelte Reduktion der GK-Expression auf einer Bindung von FoxO1 am Bindungselement oder auf einer Interaktion mit HNF-4 beruht

Effect of Homogeneous and Heterogeneous Chemical Reactions on Peristaltic Transport of a Jeffrey Fluid through a Porous Medium with Slip Condition

In this paper, the dispersion of solute matter in a Jeffrey fluid flow through a porous medium in a peristaltic channel has been investigated under the influence of slip boundary conditions. Long wavelength approximation and Taylor's limiting condition are used to obtain the average effective dispersion coefficient in both the cases of homogeneous and heterogeneous chemical reactions. The effects of various pertinent parameters on the effective dispersion coefficient are discussed. Average effective dispersion coefficient increases with amplitude ratio. That is, more dispersion in the presence of peristalsis. Further, the average effective dispersion coefficient increases with the permeability parameter and the slip parameter; but decreases with the Jeffrey number, homogeneous / heterogeneous chemical reaction rate parameter

FoxO1 and HNF-4 Are Involved in Regulation of Hepatic Glucokinase Gene Expression by Resveratrol

AbstractResveratrol, a polyphenol derived from grapes, exerts important effects on glucose and lipid metabolism, yet detailed mechanisms mediating these effects remain unknown. The liver plays a central role in energy homeostasis, and glucokinase (GK) is a key enzyme involved in glucose utilization. Resveratrol activates SIRT1 (sirtuin 1), which promotes deacetylation of the forkhead transcription factor FoxO1. Previously, we reported that FoxO1 can suppress and that HNF-4 can stimulate GK expression in the liver. Here, we examined the role of FoxO1 and HNF-4 in mediating resveratrol effects on liver GK expression. Resveratrol suppressed hepatic GK expression in vivo and in isolated hepatocytes, and knocking down FoxO1 with shRNAs disrupted this effect. Reporter gene, gel shift, supershift assay, and chromatin immunoprecipitation studies show that FoxO1 binds to the GK promoter and that the interplay between FoxO1 and HNF-4 within the GK promoter is essential for mediating the effects of resveratrol. Resveratrol promotes deacetylation of FoxO1 and enhances its recruitment to the FoxO-binding element. Conversely, resveratrol suppresses recruitment of HNF-4 to its binding site, and knockdown of FoxO1 blocks this effect of resveratrol. Coprecipitation and chromatin immunoprecipitation studies show that resveratrol enhances interaction between FoxO1 and HNF-4, reduces binding of HNF-4 to its own site, and promotes its recruitment to the FoxO site in a FoxO1-dependent manner. These results provide the first evidence that resveratrol represses GK expression via FoxO1 and that the interaction between FoxO1 and HNF-4 contributes to these effects of resveratrol.Abstract Resveratrol, a polyphenol derived from grapes, exerts important effects on glucose and lipid metabolism, yet detailed mechanisms mediating these effects remain unknown. The liver plays a central role in energy homeostasis, and glucokinase (GK) is a key enzyme involved in glucose utilization. Resveratrol activates SIRT1 (sirtuin 1), which promotes deacetylation of the forkhead transcription factor FoxO1. Previously, we reported that FoxO1 can suppress and that HNF-4 can stimulate GK expression in the liver. Here, we examined the role of FoxO1 and HNF-4 in mediating resveratrol effects on liver GK expression. Resveratrol suppressed hepatic GK expression in vivo and in isolated hepatocytes, and knocking down FoxO1 with shRNAs disrupted this effect. Reporter gene, gel shift, supershift assay, and chromatin immunoprecipitation studies show that FoxO1 binds to the GK promoter and that the interplay between FoxO1 and HNF-4 within the GK promoter is essential for mediating the effects of resveratrol. Resveratrol promotes deacetylation of FoxO1 and enhances its recruitment to the FoxO-binding element. Conversely, resveratrol suppresses recruitment of HNF-4 to its binding site, and knockdown of FoxO1 blocks this effect of resveratrol. Coprecipitation and chromatin immunoprecipitation studies show that resveratrol enhances interaction between FoxO1 and HNF-4, reduces binding of HNF-4 to its own site, and promotes its recruitment to the FoxO site in a FoxO1-dependent manner. These results provide the first evidence that resveratrol represses GK expression via FoxO1 and that the interaction between FoxO1 and HNF-4 contributes to these effects of resveratrol

High coherence superconducting microwave cavities with indium bump bonding

Low-loss cavities are important in building high-coherence superconducting quantum computers. Generating high quality joints between parts is crucial to the realization of a scalable quantum computer using the circuit quantum electrodynamics (cQED) framework. In this paper, we adapt the technique of indium bump bonding to the cQED architecture to realize high quality superconducting microwave joints between chips. We use this technique to fabricate compact superconducting cavities in the multilayer microwave integrated quantum circuits (MMIQC) architecture and achieve single photon quality factor over 300 million or single-photon lifetimes approaching 5 ms. To quantify the performance of the resulting seam, we fabricate microwave stripline resonators in multiple sections connected by different numbers of bonds, resulting in a wide range of seam admittances. The measured quality factors combined with the designed seam admittances allow us to bound the conductance of the seam at $g_\text{seam} \ge 2\times 10^{10} /(\Omega \text{m})$. Such a conductance should enable construction of micromachined superconducting cavities with quality factor of at least a billion. These results demonstrate the capability to construct very high quality microwave structures within the MMIQC architecture

Superconducting Quantum Memory with a Suspended Coaxial Resonator

A promising way to store quantum information is by encoding it in the bosonic excitations of microwave resonators. This provides for long coherence times, low dephasing rates, as well as a hardware-efficient approach to quantum error correction. There are two main methods used to make superconducting microwave resonators: traditionally machined out of bulk material, and lithographically fabricated on-chip in thin film. 3D resonators have few loss channels and larger mode volumes, and therefore smaller participations in the lossy parts, but it can be challenging to reach high material qualities. On-chip resonators can use low-loss thin films, but confine the field more tightly, resulting in higher participations and additional loss channels from the dielectric substrate. In this work, we present a design in which a dielectric scaffold supports a thin-film conductor within a 3D package, thus combining the low surface participations of bulk-machined cavities with the high quality and control over materials of thin-film circuits. By incorporating a separate chip containing a transmon qubit, we realize a quantum memory and measure single-photon lifetimes in excess of a millisecond. This hybrid 3D architecture has several advantages for scaling, as it relaxes the importance of the package and permits modular construction with separately-replaceable qubit and resonator devices.Comment: 11 pages, 8 figure

HIV-1 Dynamics in the Host Cell: A Review of Viral- and Host- Protein Interactions and Potential Therapeutic Targets for HIV-1 Infection

HIV-1, the causative agent of AIDS, is a sophisticated retrovirus that has both evolved to invade the complex human immune system and adapted to utilize the host machinery for its own propagation. A dynamic interaction between the virus and host systems can be observed at every step of the HIV-1 lifecycle. Host factors are involved not only in mounting antiviral responses, but are also hijacked by the virus to enhance viral replication. Host factors are necessary for viral replication during entry, reverse transcription, nuclear import, integration, transcription, nuclear export, translation, assembly, and budding. Recently, a new class of host factors, called “host restriction factors,” has been identified that prevent retroviral replication in a specific host cell environment and constitute an important part of intracellular innate immunity against the virus. These restriction factors act as barriers to retroviral replication at various stages within the infected cell. Nevertheless, the HIV-1 virus has learned to subvert these antiviral responses and successfully propagate within the permissive host environment. This review article describes the identification and mechanism of action of several pro- and anti-HIV-1 host factors. It is likely that we are only beginning to get a glimpse of an ongoing complex battle between HIV-1 and the host, the understanding of which should provide valuable information for the development of novel therapeutic strategies against HIV-1.