Finite Element Plastic Limit Loads of Complex Cracks in Pipes With Two-Layered Materials.

Based on the detailed three-dimensional (3D) finite element (FE) limit analyses, the present study investigates the plastic limit loads of complex-cracked pipes with two-layered materials for determining maximum load-carrying capacity or critical crack length of pipes with two-layered materials. The complex cracks in pipes with two-layered materials consist of a partial through-wall crack and 360-deg circumferential surface crack in the inner side of pipe in the same plane in pipe, which could be developed in the preemptive weld overlay region on the dissimilar metal weld (DMW) of nuclear pipe. In terms of FE limit analyses for complex-cracked pipes with two-layered materials, total thickness of pipe, depth of 360-deg internal surface crack, length of partial through-wall crack and the effect of strength mismatch between two materials are systematically considered in the present study. As for loading conditions, axial tension, global bending moment, and internal pressure are employed in the present FE analyses, and then, the confidence of the present FE procedure is confirmed by comparing the FE results with the existing solutions for complex cracks in single material. The results of the present FE plastic limit loads are compared with the existing solutions for complex-cracked pipes with two-layered materials. Also, a simple approach using equivalent single material based on the weighted average concept instead of using the properties of two materials is suggested for predicting plastic limit loads of two-layered materials. The present results can be applied to leak-before-break (LBB) analyses of nuclear piping with weld overlay.

Tumor necrosis factor-α downregulates the REIC/Dkk-3 tumor suppressor gene in normal human skin keratinocytes.

Our previous studies revealed that REIC/Dkk-3 was expressed various tissues, including skin keratinocytes. The aim of the present study was to identify the factors that regulate the expression of the dickkopf Wnt signaling pathway inhibitor 3 (REIC/Dkk­3) tumor suppressor gene in normal human skin keratinocytes (NHKs). Several growth factors and cytokines that have previously been reported to be involved in the growth and differentiation of keratinocytes were screened as potential regulators. Western blot analysis was performed using protein from NHKs cultured with/without various factors including the epidermal growth factor, tumor necrosis factor­α, transforming growth factor­ß, interleukin (IL)­1F9, IL­6, IL­8 and Ca2+. The results indicated that only TNF­α downregulated REIC/Dkk­3 expression in NHKs. Subsequently, TNF­α was confirmed to reduce the expression levels of REIC/Dkk­3 in mouse skin tissue and hair culture models. TNF­α­mediated downregulation of REIC/Dkk­3 expression in NHKs was abrogated by the addition of a TNF­α­specific antibody. In conclusion, the results indicate that TNF­α downregulates REIC/Dkk­3 expression in normal skin keratinocytes.

Expression of tumor suppressor REIC/Dkk-3 by a newly improved adenovirus vector with insertion of a hTERT promoter at the 3'-side of the transgene.

Reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3) overexpression, induced using an adenovirus (Ad)-REIC, has been revealed to have a dramatic therapeutic effect on multiple types of cancer. To achieve an improved therapeutic effect from Ad-REIC on cancer, our group previously developed an enhanced gene expression system, the C-TSC cassette [cytomegalovirus (CMV)-RU5' located upstream (C); another promoter unit composed of triple tandem promoters, human telomerase reverse transcriptase (hTERT), simian virus 40 and CMV, located downstream of the cDNA (TSC); plus a polyadenylation (polyA) signal]. When applied to the conventional Ad-REIC, this novel system induced the development of an enhanced product, Ad-C-TSC-REIC, which exhibited a noticeable anticancer effect. However, there were difficulties in terms of Ad-C-TSC-REIC productivity in HEK293 cells, which are a widely used donor cell line for viral production. Productivity of Ad-C-TSC-REIC was significantly reduced compared with the conventional Ad-REIC, as the Ad-C-TSC-REIC had a significantly higher ability to induce apoptotic cell death of not only various types of cancer cell, but also HEK293 cells. The present study aimed to overcome this problem by modifying the C-TSC structure, resulting in an improved candidate: A C-T cassette (C: CMV-RU5' located upstream; T: another promoter unit composed of a single hTERT promoter, located downstream of the cDNA plus a polyA signal), which demonstrated gene expression comparable to that of the C-TSC system. The improved adenovirus REIC/Dkk-3 product with the C-T cassette, named Ad-C-T-REIC, exhibited a higher expression level of REIC/Dkk3, similar to that of Ad-C-TSC-REIC. Notably, the vector mitigated the cell death of donor HEK293 cells, resulting in a higher rate of production of its adenovirus. These results indicated that Ad-C-T-REIC has the potential to be a useful tool for application in cancer gene therapy.

Robust cancer-specific gene expression by a novel cassette with hTERT and CMV promoter elements.

We developed and validated a novel hTERT/CMV promoter element-driven gene expression cassette that can robustly enhance cancer-specific gene expression. The following gene expressional elements were located in tandem within the plasmid construct: [hTERT core promoter, cytomegalovirus (CMV) minimized promoter, RU5' sequence, an inserted gene, BGH polyA, hTERT enhancer]; this is hereafter referred to as the hT/Cm-R-hT construct. Using various human cancer cell lines and normal cells, the cancer-specific transcription of the green fluorescent protein (GFP) gene was examined by western blotting and fluorescence microscopy. Cancer-specific gene expression was robustly achieved in the hT/Cm-R-hT plasmid in comparison to the other control hT/Cm-driven construct. Notably, the expression level of GFP observed in the hT/Cm-R-hT-driven construct was superior to that of the control plasmid with the conventional CMV promoter in HEK293 cells, which are known to possess higher hTERT activity than normal cells. We next examined the availability of hT/Cm-R-hT in detecting the target GFP expressing cancer cells from human peripheral blood mononuclear cells (PBMCs). The hT/Cm-R-hT plasmid successfully induced cancer-specific gene expression; the robust expression of GFP was observed in target HeLa cancer cells, whereas GFP was not visibly expressed in normal PBMCs. The plasmid allowed for the selective visualization of viable HeLa cancer cells in mixed cell cultures containing up to 10000-fold more PBMCs. These findings indicate that the hT/Cm-R-hT expressional system is a valuable tool for detecting viable cancer cells mixed with normal cells. The current system can therefore be applied to the in vitro detection of cancer cells that are disseminated in the blood and other types of body fluid in vivo. Since the current system can also be applied to other types of vectors, including virus vectors, this approach using the hTERT promoter-based construct is expected to become a valuable tool for enhancing cancer-specific gene expression.

Development of Lateral Flow Assay Based on Size-Controlled Gold Nanoparticles for Detection of Hepatitis B Surface Antigen.

In this study, we developed lateral flow assay (LFA) biosensors for the detection of hepatitis B surface antigens using well-controlled gold nanoparticles (AuNPs). To enhance colorimetric signals, a seeded growth method was used for the preparation of size-controlled AuNPs with a narrow size distribution. Different sizes of AuNPs in the range of 342-137.8 nm were conjugated with antibodies and then optimized for the efficient detection of LFA biosensors. The conjugation stability was investigated by UV-vis spectroscopy of AuNP dispersion at various pH values and concentrations of antibody. Based on optimized conjugation conditions, the use of 42.7 ± 0.8 nm AuNPs exhibited superior performance for the detection of LFAs relative to other sizes of AuNPs.

Identification of an S100A8 Receptor Neuroplastin-ß and its Heterodimer Formation with EMMPRIN.

We previously reported a positive feedback loop between S100A8/A9 and proinflammatory cytokines mediated by extracellular matrix metalloproteinase inducer, an S100A9 receptor. Here, we identify neuroplastin-ß as an unreported S100A8 receptor. Neuroplastin-ß and extracellular matrix metalloproteinase inducer form homodimers and a heterodimer, and they are co-localized on the surface of cultured normal human keratinocytes. Knockdown of both receptors suppressed cell proliferation and proinflammatory cytokine induction. Upon stimulation with S100A8, neuroplastin-ß recruited GRB2 and activated extracellular signal-regulated kinase, resulting in keratinocyte proliferation. Keratinocyte proliferation in response to inflammatory stimuli was accelerated in involucrin promoter-driven S100A8 transgenic mice. Further, S100A8 and S100A9 were strongly up-regulated and co-localized in lesional skin of atopic dermatitis patients. Our results indicate that neuroplastin-ß and extracellular matrix metalloproteinase inducer form a functional heterodimeric receptor for S100A8/A9 heterodimer, followed by recruitment of specific adaptor molecules GRB2 and TRAF2, and this signaling pathway is involved in activation of both keratinocyte proliferation and skin inflammation in atopic skin. Suppression of this pathway might have potential for treatment of skin diseases associated with chronic inflammation such as atopic dermatitis.

Active Secretion of Dimerized S100A11 Induced by the Peroxisome in Mesothelioma Cells.

S100A11, a small Ca2+ binding protein, acts extracellularly as a mediator of cancer progression. That raises the question of how a protein that lacks the classical secretory signal is able to be secreted outside cells without being damaged. Some insights into this question have been obtained, and there has been accumulating evidence indicating a pivotal role of a non-classical vesicle-mediated pathway using lysosomes or peroxisomes for the protein secretion. To obtain a more precise insight into the secretory mechanism of S100A11, we first screened representative cancer cells exhibiting significantly active secretion of S100A11. From the results of profiling, we turned our attention to aggressive cancer mesothelioma cells. In mesothelioma cells, we found that abundant dimeric S100A11 was produced selectively in the peroxisome after transportation of monomeric S100A11 through an interaction with PEX14, a peroxisome membrane protein, resulting in peroxisomal secretion of dimerized S100A11. In an extracellular environment in vitro, dimerized S100A11 promoted mesothelial cell invasion indirectly with the help of fibroblast cells. Overall, the results indicate that the peroxisome functions as an essential vesicle for the production of dimerized S100A11 and the subsequent secretion of the protein from mesothelioma cells and that peroxisome-mediated secretion of dimerized S100A11 might play a critical role in mesothelioma progression in a tumor microenvironment.

NRF2 Regulates PINK1 Expression under Oxidative Stress Conditions.

Mutations of the PTEN-induced putative kinase 1 (PINK1) gene are a cause of autosomal recessive forms of Parkinson's disease. Recent studies have revealed that PINK1 is an essential factor for controlling mitochondrial quality, and that it protects cells from oxidative stresses. Although there has been considerable progress in the elucidation of various aspects of PINK1 protein regulation such as activation, stability and degradation, the transcriptional regulation of PINK1 mRNA under stress conditions remains unclear. In this study, we found that nuclear factor (erythroid-derived 2)-like 2 (NRF2), an antioxidant transcription factor, regulates PINK1 expression under oxidative stress conditions. Damaged mitochondria arising from stress conditions induced NRF2-dependent transcription of the PINK1 gene through production of reactive oxygen species (ROS). Either an ROS scavenger or forced expression of KEAP1, a potent inhibitory partner to NRF2, restricted PINK1 expression induced by activated NRF2. Transcriptionally up-regulated PINK1 diminished oxidative stress-associated cell death. The results indicate that PINK1 expression is positively regulated by NRF2 and that the NRF2-PINK1 signaling axis is deeply involved in cell survival.

Exome sequencing of desmoplastic melanoma identifies recurrent NFKBIE promoter mutations and diverse activating mutations in the MAPK pathway.

Desmoplastic melanoma is an uncommon variant of melanoma with sarcomatous histology, distinct clinical behavior and unknown pathogenesis. We performed low-coverage genome and high-coverage exome sequencing of 20 desmoplastic melanomas, followed by targeted sequencing of 293 genes in a validation cohort of 42 cases. A high mutation burden (median of 62 mutations/Mb) ranked desmoplastic melanoma among the most highly mutated cancers. Mutation patterns strongly implicate ultraviolet radiation as the dominant mutagen, indicating a superficially located cell of origin. Newly identified alterations included recurrent promoter mutations of NFKBIE, encoding NF-κB inhibitor ɛ (IκBɛ), in 14.5% of samples. Common oncogenic mutations in melanomas, in particular in BRAF (encoding p.Val600Glu) and NRAS (encoding p.Gln61Lys or p.Gln61Arg), were absent. Instead, other genetic alterations known to activate the MAPK and PI3K signaling cascades were identified in 73% of samples, affecting NF1, CBL, ERBB2, MAP2K1, MAP3K1, BRAF, EGFR, PTPN11, MET, RAC1, SOS2, NRAS and PIK3CA, some of which are candidates for targeted therapies.

Phylogenetic analyses of melanoma reveal complex patterns of metastatic dissemination.

Melanoma is difficult to treat once it becomes metastatic. However, the precise ancestral relationship between primary tumors and their metastases is not well understood. We performed whole-exome sequencing of primary melanomas and multiple matched metastases from eight patients to elucidate their phylogenetic relationships. In six of eight patients, we found that genetically distinct cell populations in the primary tumor metastasized in parallel to different anatomic sites, rather than sequentially from one site to the next. In five of these six patients, the metastasizing cells had themselves arisen from a common parental subpopulation in the primary, indicating that the ability to establish metastases is a late-evolving trait. Interestingly, we discovered that individual metastases were sometimes founded by multiple cell populations of the primary that were genetically distinct. Such establishment of metastases by multiple tumor subpopulations could help explain why identical resistance variants are identified in different sites after initial response to systemic therapy. One primary tumor harbored two subclones with different oncogenic mutations in CTNNB1, which were both propagated to the same metastasis, raising the possibility that activation of wingless-type mouse mammary tumor virus integration site (WNT) signaling may be involved, as has been suggested by experimental models.

Construction and characterization of Cu²âº, Ni²âº, Zn²âº, and Co²âº modified-DNA crystals.

We studied the physical characteristics of modified-DNA (M-DNA) double crossover crystals fabricated via substrate-assisted growth with various concentrations of four different divalent metallic ions, Cu(2+), Ni(2+), Zn(2+), and Co(2+). Atomic force microscopy (AFM) was used to test the stability of the M-DNA crystals with different metal ion concentrations. The AFM images show that M-DNA crystals formed without deformation at up to the critical concentrations of 6 mM of [Cu(2+)], 1.5 mM of [Ni(2+)], 1 mM of [Zn(2+)], and 1 mM of [Co(2+)]. Above these critical concentrations, the M-DNA crystals exhibited deformed, amorphous structures. Raman spectroscopy was then used to identify the preference of the metal ion coordinate sites. The intensities of the Raman bands gradually decreased as the concentration of the metal ions increased, and when the metal ion concentrations increased beyond the critical values, the Raman band of the amorphous M-DNA was significantly suppressed. The metal ions had a preferential binding order in the DNA molecules with G-C and A-T base pairs followed by the phosphate backbone. A two-probe station was used to measure the electrical current-voltage properties of the crystals which indicated that the maximum currents of the M-DNA complexes could be achieved at around the critical concentration of each ion. We expect that the functionalized ion-doped M-DNA crystals will allow for efficient devices and sensors to be fabricated in the near future.

Erratum to: Modeling precision treatment of breast cancer.

During the type-setting of the final version of the article [1] some of the additional files were swapped. The correct files are republished in this Erratum.

Practical approach to determine sample size for building logistic prediction models using high-throughput data.

An empirical method of sample size determination for building prediction models was proposed recently. Permutation method which is used in this procedure is a commonly used method to address the problem of overfitting during cross-validation while evaluating the performance of prediction models constructed from microarray data. But major drawback of such methods which include bootstrapping and full permutations is prohibitively high cost of computation required for calculating the sample size. In this paper, we propose that a single representative null distribution can be used instead of a full permutation by using both simulated and real data sets. During simulation, we have used a dataset with zero effect size and confirmed that the empirical type I error approaches to 0.05. Hence this method can be confidently applied to reduce overfitting problem during cross-validation. We have observed that pilot data set generated by random sampling from real data could be successfully used for sample size determination. We present our results using an experiment that was repeated for 300 times while producing results comparable to that of full permutation method. Since we eliminate full permutation, sample size estimation time is not a function of pilot data size. In our experiment we have observed that this process takes around 30min. With the increasing number of clinical studies, developing efficient sample size determination methods for building prediction models is critical. But empirical methods using bootstrap and permutation usually involve high computing costs. In this study, we propose a method that can reduce required computing time drastically by using representative null distribution of permutations. We use data from pilot experiments to apply this method for designing clinical studies efficiently for high throughput data.

Transcription restores DNA repair to heterochromatin, determining regional mutation rates in cancer genomes.

Somatic mutations in cancer are more frequent in heterochromatic and late-replicating regions of the genome. We report that regional disparities in mutation density are virtually abolished within transcriptionally silent genomic regions of cutaneous squamous cell carcinomas (cSCCs) arising in an XPC(-/-) background. XPC(-/-) cells lack global genome nucleotide excision repair (GG-NER), thus establishing differential access of DNA repair machinery within chromatin-rich regions of the genome as the primary cause for the regional disparity. Strikingly, we find that increasing levels of transcription reduce mutation prevalence on both strands of gene bodies embedded within H3K9me3-dense regions, and only to those levels observed in H3K9me3-sparse regions, also in an XPC-dependent manner. Therefore, transcription appears to reduce mutation prevalence specifically by relieving the constraints imposed by chromatin structure on DNA repair. We model this relationship among transcription, chromatin state, and DNA repair, revealing a new, personalized determinant of cancer risk.

Personalized identification of altered pathways in cancer using accumulated normal tissue data.

MOTIVATION: Identifying altered pathways in an individual is important for understanding disease mechanisms and for the future application of custom therapeutic decisions. Existing pathway analysis techniques are mainly focused on discovering altered pathways between normal and cancer groups and are not suitable for identifying the pathway aberrance that may occur in an individual sample. A simple way to identify individual's pathway aberrance is to compare normal and tumor data from the same individual. However, the matched normal data from the same individual are often unavailable in clinical situation. Therefore, we suggest a new approach for the personalized identification of altered pathways, making special use of accumulated normal data in cases when a patient's matched normal data are unavailable. The philosophy behind our method is to quantify the aberrance of an individual sample's pathway by comparing it with accumulated normal samples. We propose and examine personalized extensions of pathway statistics, overrepresentation analysis and functional class scoring, to generate individualized pathway aberrance score. RESULTS: Collected microarray data of normal tissue of lung and colon mucosa are served as reference to investigate a number of cancer individuals of lung adenocarcinoma (LUAD) and colon cancer, respectively. Our method concurrently captures known facts of cancer survival pathways and identifies the pathway aberrances that represent cancer differentiation status and survival. It also provides more improved validation rate of survival-related pathways than when a single cancer sample is interpreted in the context of cancer-only cohort. In addition, our method is useful in classifying unknown samples into cancer or normal groups. Particularly, we identified 'amino acid synthesis and interconversion' pathway is a good indicator of LUAD (Area Under the Curve (AUC) 0.982 at independent validation). Clinical importance of the method is providing pathway interpretation of single cancer, even though its matched normal data are unavailable. AVAILABILITY AND IMPLEMENTATION: The method was implemented using the R software, available at our Web site: http://bibs.snu.ac.kr/ipas. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DNAX-activating protein 10 (DAP10) membrane adaptor associates with receptor for advanced glycation end products (RAGE) and modulates the RAGE-triggered signaling pathway in human keratinocytes.

The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of many inflammatory, degenerative, and hyperproliferative diseases, including cancer. Previously, we revealed mechanisms of downstream signaling from ligand-activated RAGE, which recruits TIRAP/MyD88. Here, we showed that DNAX-activating protein 10 (DAP10), a transmembrane adaptor protein, also binds to RAGE. By artificial oligomerization of RAGE alone or RAGE-DAP10, we found that RAGE-DAP10 heterodimer formation resulted in a marked enhancement of Akt activation, whereas homomultimeric interaction of RAGE led to activation of caspase 8. Normal human epidermal keratinocytes exposed to S100A8/A9, a ligand for RAGE, at a nanomolar concentration mimicked the pro-survival response of RAGE-DAP10 interaction, although at a micromolar concentration, the cells mimicked the pro-apoptotic response of RAGE-RAGE. In transformed epithelial cell lines, A431 and HaCaT, in which endogenous DAP10 was overexpressed, and S100A8/A9, even at a micromolar concentration, led to cell growth and survival due to RAGE-DAP10 interaction. Functional blocking of DAP10 in the cell lines abrogated the Akt phosphorylation from S100A8/A9-activated RAGE, eventually leading to an increase in apoptosis. Finally, S100A8/A9, RAGE, and DAP10 were overexpressed in the psoriatic epidermis. Our findings indicate that the functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated survival and/or apoptotic response of keratinocytes.

Fully automated circulating tumor cell isolation platform with large-volume capacity based on lab-on-a-disc.

Full automation with high purity for circulating tumor cell (CTC) isolation has been regarded as a key goal to make CTC analysis a "bench-to-bedside" technology. Here, we have developed a novel centrifugal microfluidic platform that can isolate the rare cells from a large volume of whole blood. To isolate CTCs from whole blood, we introduce a disc device having the biggest sample capacity as well as manipulating blood cells for the first time. The fully automated disc platform could handle 5 mL of blood by designing the blood chamber having a triangular obstacle structure (TOS) with lateral direction. To guarantee high purity that enables molecular analysis with the rare cells, CTCs were bound to the microbeads covered with anti-EpCAM to discriminate density between CTCs and blood cells and the CTCs being heavier than blood cells were only settled under a density gradient medium (DGM) layer. To understand the movement of CTCs under centrifugal force, we performed computational fluid dynamics simulation and found that their major trajectories were the boundary walls of the DGM chamber, thereby optimizing the chamber design. After whole blood was inserted into the blood chamber of the disc platform, size- and density-amplified cancer cells were isolated within 78 min, with minimal contamination as much as approximately 12 leukocytes per milliliter. As a model of molecular analysis toward personalized cancer treatment, we performed epidermal growth factor receptor (EGFR) mutation analysis with HCC827 lung cancer cells and the isolated cells were then successfully detected for the mutation by PCR clamping and direct sequencing.

Anti-cancer effects of REIC/Dkk-3-encoding adenoviral vector for the treatment of non-small cell lung cancer.

OBJECTIVES: REIC/Dkk-3 is down-regulated in a broad range of human cancer cells and is considered to function as a tumor suppressor. We previously reported that REIC/Dkk-3-expressing adenovirus vector (Ad-REIC) induced endoplasmic reticulum (ER) stress and cancer-specific apoptosis in human prostate cancer. In this study, we examined the therapeutic impact of Ad-REIC on non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: We examined the anti-tumor effect of Ad-REIC on 25 NSCLC cell lines in vitro and A549 cells in vivo. Two of these cell lines were artificially established as EGFR-tyrosine kinase inhibitor (TKI) resistant sublines. RESULTS: Ad-REIC-treatment inhibited the cell viability by 40% or more in 13 (52%) of the 25 cell lines at multiplicity of infection (MOI) of 20 (20 MOI). These cell lines were regarded as being highly sensitive cells. The cell viability of a non-malignant immortalized cell line, OUMS-24, was not inhibited at 200 MOI of Ad-REIC. The effects of Ad-REIC on EGFR-TKI resistant sublines were equivalent to those in the parental cell lines. Here, we demonstrated that Ad-REIC treatment activated c-Jun N-terminal kinase (JNK) in NSCLC cell lines, indicating the induction of ER stress with GRP78/BiP (GRP78) up-regulation and resulting in apoptosis. A single intratumoral injection of Ad-REIC significantly inhibited the tumorigenic growth of A549 cells in vivo. As predictive factors of sensitivity for Ad-REIC treatment in NSCLC, we examined the expression status of GRP78 and coxsackievirus and adenovirus receptor (CAR). We found that the combination of the GRP78 and CAR expressional statuses may be used as a predictive factor for Ad-REIC sensitivity in NSCLC cells. CONCLUSION: Ad-REIC induced JNK activation and subsequent apoptosis in NSCLC cells. Our study indicated that Ad-REIC has therapeutic potential against NSCLC and that the expression statuses of GRP78 and CAR may predict a potential therapeutic benefit of Ad-REIC.

Dramatic increase in expression of a transgene by insertion of promoters downstream of the cargo gene.

For expression of genes in mammalian cells, various vectors have been developed using promoters including CMV, EF-1α, and CAG promoters and have been widely used. However, such expression vectors sometimes fail to attain sufficient expression levels depending on the nature of cargo genes and/or on host cell types. In the present study, we aimed to develop a potent promoter system that enables high expression levels of cargo genes ubiquitously in many different cell types. We found that insertion of an additional promoter downstream of a cargo gene greatly enhanced the expression levels. Among the constructs we tested, C-TSC cassette (C: CMV-RU5' located upstream; TSC: another promoter unit composed of triple tandem promoters, hTERT, SV40, and CMV, located downstream of the cDNA plus a polyadenylation signal) had the most potent capability, showing far higher efficiency than that of potent conventional vector systems. The results indicate that the new expression system is useful for production of recombinant proteins in mammalian cells and for application as a gene therapeutic measure.

A novel gene expression system strongly enhances the anticancer effects of a REIC/Dkk-3-encoding adenoviral vector.

Gene expression systems with various promoters, including the cytomegalovirus (CMV) promoter, have been developed to increase the gene expression in a variety of normal and cancer cells. In particular, in the clinical trials of cancer gene therapy, a more efficient and robust gene expression system is required to achieve sufficient therapeutic outcomes. By inserting the triple translational enhancer sequences of human telomerase reverse transcriptase (hTERT), Simian virus 40 (SV40) and CMV downstream of the sequence of the BGH polyA, we were able to develop a novel gene expression system that significantly enhances the expression of the genes of interest. We termed this novel gene expression cassette the super gene expression (SGE) system, and herein verify the utility of the SGE cassette for a replication-deficient adenoviral vector. We newly developed an adenoviral vector expressing the tumor suppressor, reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3), based on the CMV promoter-driven SGE system (Ad-SGE-REIC) and compared the therapeutic utility of Ad-SGE-REIC with that of the conventional adenoviral vectors (Ad-CMV-REIC or Ad-CAG-REIC). The results demonstrated that the CMV promoter-SGE system allows for more potent gene expression, and that the Ad-SGE-REIC is superior to conventional adenoviral systems in terms of the REIC protein expression and therapeutic effects. Since the SGE cassette can be applied for the expression of various therapeutic genes using various vector systems, we believe that this novel system will become an innovative tool in the field of gene expression and gene therapy.