Checkpoint kinase 1 protein expression indicates sensitization to therapy by checkpoint kinase 1 inhibition in non–small cell lung cancer

Background

When presenting with advanced stage disease, lung cancer patients have <5% 5-y survival. The overexpression of checkpoint kinase 1 (CHK1) is associated with poorer outcomes and may contribute to therapy resistance. Targeting CHK1 with small-molecule inhibitors in p53 mutant tumors might improve the effectiveness of chemotherapy and radiotherapy in non–small cell lung cancer (NSCLC).

Methods

We evaluated CHK1 messenger RNA and protein levels in multiple NSCLC cell lines. We assessed cell line sensitization to gemcitabine, pemetrexed, and radiotherapy by CHK1 inhibition with the small molecule AZD7762 using proliferation and clonogenic cell survival assays. We analyzed CHK1 signaling by Western blotting to confirm that AZD7762 inhibits CHK1.

Results

We selected two p53 mutant NSCLC cell lines with either high (H1299) or low (H1993) CHK1 levels for further analysis. We found that AZD7762 sensitized both cell lines to gemcitabine, pemetrexed, and radiotherapy. Chemosensitization levels were greater, however, for the higher CHK1 protein expressing cell line, H1299, when compared with H1993. Furthermore, analysis of the CHK1 signaling pathway showed that H1299 cells have an increased dependence on the CHK1 pathway in response to chemotherapy. There was no increased sensitization to radiation in H1299 versus H1993.

Conclusions

CHK1 inhibition by AZD7762 preferentially sensitizes high CHK1 expressing cells, H1299, to anti-metabolite chemotherapy as compared with low CHK1 expressing H1993 cells. Thus, CHK1 inhibitors may improve the efficacy of standard lung cancer therapies, especially for those subgroups of tumors harboring higher expression levels of CHK1 protein.     

Trajectories of Substance Use among Child Welfare-Involved Youth: Longitudinal Associations with Child Maltreatment History and Emotional/Behavior Problems

Background: Maltreated children experience a variety of adverse outcomes including substance use problems. Although previous research indicated that there may be distinct trajectories of substance use among these youth, studies have examined them as if they were a single homogeneous group. Objectives: The goals of this study were to explore substance use trajectories among child welfare-involved youth and to identify characteristics that distinguish substance use trajectories. Methods: Data from the National Survey of Child and Adolescent Well-Being (NSCAW II) were used. Multilevel latent growth mixture modeling (MLGMM) was performed using a subsample of 625 youth from ages 11–17 years investigated for maltreatment in 2008–2009. Measures included self-reported use of substance use during the previous 30 days, demographic characteristics, maltreatment history, placement in out-of-home care, and behavioral health problems. Results: MLGMM identified two distinct substance use trajectory classes including High Stable Substance Use and Rapid Progression Use. Findings suggest that the experience of physical abuse is the key factor that distinguishes the two groups. When the effects of class-specific covariates were examined, results suggest that involvement in substance use behavior and its escalation vary between groups and are affected by youth’s different previous experiences. Conclusions/Importance: The results have important implications for understanding individual differences in substance use behavior over time and how these differences were shaped by youth’s experiences of family adversity. Study findings may be helpful for developing and enhancing the effectiveness of interventions targeted at decreasing substance use behaviors in child welfare-involved youth.     

Association of TM4SF4 with the Human Thiamine Transporter-2 in Intestinal Epithelial Cells

Background

The human thiamine transporter-2 (hTHTR-2) is involved in the intestinal absorption of thiamine. Recent studies with membrane transporters of other nutrients/substrates have shown that they have associated proteins that affect different aspects of their physiology and cell biology. Nothing is known about protein(s) that interact with hTHTR-2 in intestinal epithelial cells and influence its physiological function and/or its cell biology.

Aims

The aim of this study was to identify protein partner(s) that interact with hTHTR-2 in human intestinal cells and determine the physiological/biological consequence of that interaction.

Methods

The yeast split-ubiquitin two-hybrid approach was used to screen a human intestinal cDNA library. GST-pull-down and cellular co-localization approaches were used to confirm the interaction between hTHTR-2 and the associated protein(s). The effect of such an interaction on hTHTR-2 function was examined by ³H-thiamine uptake assays.

Results

Our screening results identified the human TransMembrane 4 SuperFamily 4 (TM4SF4) as a potential interactor with hTHTR-2. This interaction was confirmed by an in vitro GST-pull-down assay, and by live-cell confocal imaging of HuTu-80 cells co-expressing hTHTR-2–GFP and mCherry–TM4SF4 (the latter displayed a significant overlap of these two proteins in intracellular vesicles and at the cell membrane). Co-expression of hTHTR-2 with TM4SF4 in HuTu-80 cells led to a significant induction in thiamine uptake. In contrast, silencing TM4SF4 with gene-specific siRNA led to a significant decrease in thiamine uptake.

Conclusions

These results show for the first time that the accessory protein TM4SF4 interacts with hTHTR-2 and influences the physiological function of the thiamine transporter.     

Early lesions of follicular lymphoma: a genetic perspective

The pathogenesis of follicular lymphoma is a multi-hit process progressing over many years through the accumulation of numerous genetic alterations. Besides the hallmark t(14;18), it is still unclear which other oncogenic hits contribute to the early steps of transformation and in which precursor stages these occur. To address this issue, we performed high-resolution comparative genomic hybridization microarrays on laser-capture micro-dissected cases of follicular lymphoma in situ (n=4), partial involvement by follicular lymphoma (n=4), and duodenal follicular lymphoma (n=4), assumed to represent, potentially, the earliest stages in the evolution of follicular lymphoma. Cases of reactive follicular hyperplasia (n=2), uninvolved areas from follicular lymphoma in situ lymph nodes, follicular lymphoma grade 1–2 (n=5) and follicular lymphoma grade 3A (n=5) were used as controls. Surprisingly, alterations involving several relevant (onco)genes were found in all entities, but at significantly lower proportions than in overt follicular lymphoma. While the number of alterations clearly assigns all these entities as precursors, the pattern of partial involvement by follicular lymphoma alterations was quantitatively and qualitatively closer to that of follicular lymphoma, indicating significant selective pressure in line with its faster rate of progression. Among the most notable alterations, we observed and validated deletions of 1p36 and gains of the 7p and 12q chromosomes and related oncogenes, which include some of the most recurrent oncogenic alterations in overt follicular lymphoma (TNFRSF14, EZH2, MLL2). By further delineating distinctive and hierarchical molecular and genetic features of early follicular lymphoma entities, our analysis underlines the importance of applying appropriate criteria for the differential diagnosis. It also provides a first set of candidates likely to be involved in the cascade of hits that pave the path of the various progression phases to follicular lymphoma development.     

Heterodimeric coiled-coil interactions of human GABAB receptor

Metabotropic GABA_B receptor is a G protein-coupled receptor that mediates inhibitory neurotransmission in the CNS. It functions as an obligatory heterodimer of GABA_B receptor 1 (GBR1) and GABA_B receptor 2 (GBR2) subunits. The association between GBR1 and GBR2 masks an endoplasmic reticulum (ER) retention signal in the cytoplasmic region of GBR1 and facilitates cell surface expression of both subunits. Here, we present, to our knowledge, the first crystal structure of an intracellular coiled-coil heterodimer of human GABA_B receptor. We found that polar interactions buried within the hydrophobic core determine the specificity of heterodimer pairing. Disruption of the hydrophobic coiled-coil interface with single mutations in either subunit impairs surface expression of GBR1, confirming that the coiled-coil interaction is required to inactivate the adjacent ER retention signal of GBR1. The coiled-coil assembly buries an internalization motif of GBR1 at the heterodimer interface. The ER retention signal of GBR1 is not part of the core coiled-coil structure, suggesting that it is sterically shielded by GBR2 upon heterodimer formation.The major inhibitory neurotransmitter in the CNS is GABA. Metabotropic GABA_B receptor is a G protein-coupled receptor (GPCR) that mediates slow synaptic inhibition (1, 2). It constitutes an important drug target for many neurological disorders, including epilepsy, spasticity, anxiety, and nociception (1, 2).Formation of a functional GABA_B receptor requires the heterodimeric assembly of GABA_B receptor 1 (GBR1) and GABA_B receptor 2 (GBR2) subunits (3–7). Both consist of an N-terminal extracellular domain, a seven-helix transmembrane domain, and a C-terminal intracellular domain. The intracellular domain of each subunit contains a stretch of coiled-coil sequence, and interaction between the coiled-coil helices is partly responsible for GABA_B receptor heterodimerization (5, 8).The intracellular region of GABA_B receptor hosts elements that control receptor trafficking (9). Specifically, GBR1 has a di-leucine internalization signal (EKSRLL) (9) and an endoplasmic reticulum (ER) retention signal (RSRR) (9–11) located within or near its coiled-coil domain (9). GBR1 is trapped within the ER when expressed alone (12) but can reach the cell surface upon association with GBR2 (9, 11). Mutation or removal of the ER retention signal in GBR1 results in plasma membrane expression of GBR1 (9–11). Furthermore, interaction between the coiled-coil domains of GBR1 and GBR2 masks this ER retention signal to facilitate the cell surface expression of both subunits (9–11). Although mutation of the di-leucine motif itself is not sufficient to release GBR1 from intracellular retention, it enhances cell surface expression of various GBR1 mutants that lack the ER retention signal (9).The coiled-coil domain of GBR1 associates with a number of intracellular proteins involved in trafficking, including the coat protein complex I (COPI) (13), the scaffolding protein 14-3-3 (13, 14), the GPCR interacting scaffolding protein GISP (15), and the guanidine exchange factor msec7-1 (16). In particular, COPI specifically recognizes the ER retention signal sequence of GBR1 and is involved in the intracellular retention of GBR1 (13). The msec7-1 protein increases the cell surface expression of GABA_B receptor by binding to the di-leucine internalization motif (16).Despite its important role in GABA_B receptor assembly and trafficking, the atomic details of the coiled-coil interaction between subunits are not known. In this study, we present the crystal structure of a GBR1/GBR2 coiled-coil heterodimer and identify specific contacts at the heterodimer interface that control the surface expression of GBR1.     

Enhancement of protein expression by alphavirus replicons by designing self-replicating subgenomic RNAs

Since the development of infectious cDNA clones of viral RNA genomes and the means of delivery of the in vitro-synthesized RNA into cells, alphaviruses have become an attractive system for expression of heterologous genetic information. Alphaviruses replicate exclusively in the cytoplasm, and their genetic material cannot recombine with cellular DNA. Alphavirus genome-based, self-replicating RNAs (replicons) are widely used vectors for expression of heterologous proteins. Their current design relies on replacement of structural genes, encoded by subgenomic RNAs (SG RNA), with heterologous sequences of interest. The SG RNA is transcribed from a promoter located in the alphavirus-specific RNA replication intermediate and is not further amplified. In this study, we have applied the accumulated knowledge of the mechanism of alphavirus replication and promoter structures, in particular, to increase the expression level of heterologous proteins from Venezuelan equine encephalitis virus (VEEV)-based replicons. During VEEV infection, replication enzymes are produced in excess to RNA replication intermediates, and a large fraction of them are not involved in RNA synthesis. The newly designed constructs encode SG RNAs, which are not only transcribed from the SG promoter, but are additionally amplified by the previously underused VEEV replication enzymes. These replicons produce SG RNAs and encoded proteins of interest 10- to 50-fold more efficiently than those using a traditional design. A modified replicon encoding West Nile virus (WNV) premembrane and envelope proteins efficiently produced subviral particles and, after a single immunization, elicited high titers of neutralizing antibodies, which protected mice from lethal challenge with WNV.Alphaviruses are a group of enveloped viruses with a positive-strand RNA genome that replicate in most commonly used cell lines to titers exceeding 10¹⁰ infectious units (inf.u)/mL (1, 2). Upon infection, the genomic RNA serves as a template for translation of viral nonstructural proteins that form replication complexes (3). Within a few hours postinfection, these complexes synthesize large amounts of viral genomic and subgenomic (SG) RNA (3). The SG RNA is transcribed from the SG promoter and serves as a template for translation of viral structural proteins: capsid, E2 and E1, which ultimately assemble with genomic RNA into infectious viral particles. This highly efficient virus-specific RNA and protein synthesis, coupled with the availability of infectious cDNA clones, have made alphaviruses an attractive system for designing self-replicating vectors for delivery and expression of heterologous genetic information. The most widely used alphavirus-based expression systems are based on replacement of viral structural genes by a gene(s) of interest (4). These modified viral genomes, termed replicons, can be synthesized in vitro and delivered into cells either by transfection or in infectious viral particles, which deliver essentially every packaged RNA molecule into the cells both in vivo and in vitro.In recent years, significant progress has been made in our understanding of the mechanism of alphavirus replication. Detailed studies have elucidated the structure and function of the RNA promoters, critical aspects of virus–host cell interactions, and the composition of the replication complexes (5–12). These mechanistic studies of alphavirus replication raised the question of whether we are using their entire expression potential, and whether the traditional replicon design can be further improved to achieve higher levels of heterologous protein production. In this project, we sought to apply the latest advances in understanding of alphavirus RNA replication to design a new generation of Venezuelan equine encephalitis virus (VEEV) genome-based expression systems. The distinguishing feature of these constructs is the modification of the SG RNAs. These SG RNAs have been engineered to contain the cis-acting promoter elements, which are normally present at the 5′ end of the viral genome and mediate genomic RNA replication (8, 13, 14). Thus, in these newly designed VEEV replicons (VEErep), the SG RNAs were not only transcribed from the SG promoter, but were capable of replication/amplification by the VEEV replication complexes. As a result, the heterologous gene expression was more efficient than that of the existing constructs, which use replicons with the standard SG RNAs. The expression level of heterologous protein encoded by the improved replicons was also found to be dependent on coexpression of VEEV capsid protein. The VEEV replicons, which use both amplification of the SG RNA and express capsid protein, provide a platform for development of a variety of more efficient expression systems and have numerous applications. To illustrate this, we have generated a VEEV replicon encoding the premembrane and envelope (prM/E) proteins of West Nile virus (WNV). Particles containing the newly designed replicons induced high levels of WNV E protein expression in vitro and elicited robust protective immunity in mice.     

Nano-architecture of silica nanoparticles as a tool to tune both electrochemical and catalytic behavior of NiII@SiO2

The present work introduces a facile synthetic route for efficient doping of [Ni^II(bpy)_x] into silica nanoparticles with various sizes and architectures. Variation of the latter results in different concentrations of the Ni^II complexes at the interface of the composite nanoparticles. The UV-Vis analysis of the nanoparticles reveals changes in the inner-sphere environment of the Ni^II complexes when embedded into the nanoparticles, while the inner-sphere of Ni^II is invariant for the nanoparticles with different architecture. Comparative analysis of the electrochemically generated redox transformations of the Ni^II complexes embedded in the nanoparticles of various architectures reveals the latter as the main factor controlling the accessibility of Ni^II complexes to the redox transitions which, in turn, controls the electrochemical behavior of the nanoparticles. The work also highlights an impact of the nanoparticulate architecture in catalytic activity of the Ni^II complexes within the different nanoparticles in oxidative C–H fluoroalkylation of caffeine. Both low leakage and high concentration of the Ni^II complexes at the interface of the composite nanoparticles enables fluoroalkylated caffeine to be obtained in high yields under recycling of the nanocatalyst five times at least.

The present work introduces a facile synthetic route for efficient doping of [Ni^II(bpy)_x] into silica nanoparticles with various sizes and architectures.     

Molecular and biochemical study of glutaric aciduria type 1 in 49 Russian families: nine novel mutations in the GCDH gene

Metabolic Brain Disease - Glutaric aciduria type 1 (GA1, deficiency of glutaryl CoA dehydrogenase, glutaric acidemia type 1) (ICD-10 code: E72.3; MIM 231670) is an autosomal recessive disease...