Reverse Transfection Research Articles

Nanofiber-mediated microRNA delivery to enhance differentiation and maturation of oligodendroglial precursor cells

Remyelination in the central nervous system (CNS) is critical in the treatment of many neural pathological conditions. Unfortunately, the ability to direct and enhance oligodendrocyte (OL) differentiation and maturation remains limited. It is known that microenvironmental signals, such as substrate topography and biochemical signaling, regulate cell fate commitment. Therefore, in this study, we developed a nanofiber-mediated microRNA (miR) delivery method to control oligodendroglial precursor cell (OPC) differentiation through a combination of fiber topography and gene silencing. Using poly(ε-caprolactone) nanofibers, efficient knockdown of OL differentiation inhibitory regulators were achieved by either nanofiber alone (20–40%, p<0.05) or the synergistic integration with miR-219 and miR-338 (up to 60%, p<0.05). As compared to two-dimensional culture, nanofiber topography enhanced OPC differentiation by inducing 2-fold increase in RIP+ cells (p<0.01) while the presence of miRs further enhanced the result to 3-fold (p<0.001). In addition, nanofiber-mediated delivery of miR-219 and miR-338 promoted OL maturation by increasing the number of MBP+ cells significantly (p<0.01). Taken together, the results demonstrate the efficacy of nanofibers in providing topographical cues and microRNA reverse transfection to direct OPC differentiation. Such scaffolds may find useful applications in directing oligodendrocyte differentiation and myelination for treatment of CNS pathological conditions that require remyelination.

Dissecting the invasive potential of esophageal cancer by siRNA library screening.

86 Background: The low survival for esophageal cancer is in part attributed to its high invasive potential and distant metastasis. In cancer, abnormal cell migration is an essential component of metastasis, and it is reasonable to consider that the conversion between different forms of morphology permits tumor cells to metastasise. Discovering regulators of esophageal cancer morphogenesis may aid in the development of novel targeted therapies that limit metastatic potential. Methods: GOhTRT cells were seeded and treated with siRNA (Human Druggable Genome, Dharmacon) by reverse transfection. Cells were fixed, immunostained for DNA, tubulin and actin and imaged with automated microscope. Cell images were processed using the InCell Analyzer software and the R statistical software systems CellHTS2 and RNAither. The effect of RNAi knockdown on cell viability and cell motility were assessed using MTT cell proliferation assay and scratch wound assay. Results: 127 gene candidates greatly exhibited effects on F-actin and α-tubulin area staining. This list was refined to six high quality candidates (RRM2, ITGB8, GPS1, SPRY1, NOL1 and MYO9B). Silencing of GPS1, MYO9B and SPRY1 increased the rate of migration in a scratch wound assay, with 86.98% ± 3.097%, 75.78% ±5.454% and 72.97% ± 5.463% (p =0.0022) respectively. There was no significant difference in cell viability absorbance values for siGPS1 (0.9037 ± 0.06575; p =0.1905) and siSPRY1 (0.9088 ± 0.09849; p =0.2985), which suggests that the increased rate of wound closure previously seen is by virtue of migratory signalling as oppose to an increase in cell proliferation. Cell viability was decreased considerably in siRRM2 cells (0.2492 ± 0.02798; p &lt;0.0001) and siMYO9B (0.4048 ± 0.04663; p&lt;0.0001) in comparison to siNT cells (1.046 ± 0.07712). Conclusions: In summary, this screen successfully identified high confidence hits associated with cytoskeletal remodelling, some of which are already associated with metastasis in literature and database searches. Further mechanistic studies and gene pathway analysis of candidate genes will provide novel therapeutic targets which can be utilised to block the spread of cancer in patients.

T5 Opg Regulates Pulmonary Arterial Smooth Muscle Cell Proliferation And The Expression Of Pah-associated Genes Via Fas

Introduction and objectives Pulmonary arterial hypertension (PAH) is a fatal lung disease characterised by progressive pulmonary vascular remodelling, a key component of which is the proliferation and migration of pulmonary arterial smooth muscle cells (PA-SMCs). Although current therapies are good at alleviating symptoms, they do not reverse the underlying pulmonary vascular remodelling. We have previously demonstrated that the secreted glycoprotein, osteoprotegerin (OPG, TNFRSF11B), is elevated within pulmonary vascular lesions and serum from idiopathic PAH (IPAH) patients and induces PA-SMC proliferation and migration in vitro . Furthermore, genetic deletion or antibody blockade of OPG can prevent and reverse disease in pre-clinical animal models. However, how OPG signals to mediate PA-SMC phenotype remains unclear. We therefore aimed to characterise the OPG signalling cascade in PA-SMCs, and identify the receptor through which this is mediated. Methods PA-SMCs were stimulated with 0.2% FCS and OPG (50 ng/ml) for 10 and 60 min. Phosphorylation targets were identified by Kinex antibody microarray (Kinexus, Canada). An RNA expression microarray (Agilent) was performed on PA-SMCs following 6-hour OPG stimulation. OPG binding partners were identified following reverse transfection of HEK293 cells with 2054 human membrane proteins (Retrogenix, Sheffield, UK). Interactions were confirmed in PA-SMCs by co-immunoprecipitation. PA-SMCs were pre-treated with Fas neutralising antibody (1500 ng/ml), TRAIL antibody (1500 ng/ml) or both antibodies, 30 min before OPG stimulation. Proliferation was assessed after 72 h. Results OPG induced significant activation of CDK4 and CDK5, HSP27 and ERK1/2, and significant decrease in phospho-mTOR. OPG significantly altered the expression of 57 PAH-associated genes, including TRAIL. Four novel OPG interactions with IL1RAcP, Fas, TMPRSS11D and GAP43 were identified and we confirmed OPG interaction with IL1RAcP and Fas in PA-SMC. Fas RNA expression was elevated in IPAH PA-SMCs and protein expression was elevated in the right ventricle and pulmonary artery from IPAH patients. Fas blockade reduced OPG-induced proliferation by ~40%, which was further reduced by simultaneous TRAIL blockade. Fas blockade also prevented OPG-induced PDGFRA and TNC RNA expression. Conclusions These studies begin to reveal the intracellular signalling mechanisms and receptor through which OPG induces PA-SMC proliferation, further highlighting the therapeutic potential of targeting OPG in PAH.

Abstract 4371: Tumor suppressive microRNA-218 inhibits cancer cell migration and invasion via targeting LASP1 in prostate cancer

Abstract BACKGROUND: Prostate cancer (PCa) is the most frequently diagnosed cancer and the second leading cause of cancer death among men in developed countries. Disease progression and the development of hormone refractory disease remain major cause of cancer-related death.Understanding the molecular mechanisms of PCa progression and metastatic pathways using currently available genomic approaches would help to improve therapies for and prevention of the disease. Our recent study of microRNA (miRNA) expression signature of PCa has revealed that microRNA-218 (miR-218) was significantly downregulated in cancer tissues, suggesting that miR-218 may be a putative tumor-suppressive miRNA in PCa. The aim of this study was to investigate the functional significance of miR-218 in PCa and to identify novel miR-218-regulated cancer pathways and target genes involved in PCa oncogenesis and metastasis.METHODS: The expression levels of miR-218 were validated using real-time RT-PCR using PCa clinical specimens and cell lines (PC3 and DU145). Cell proliferation, migration and invasion assays were performed to investigate the functional significance of miR-218 and its target gene, LIM and SH3 protein 1 (LASP1) in PCa cell lines.Cells were transfected with mature miR-218 or si-LASP1 by reverse transfection methods. Genome-wide gene expression data and in silico analysis were used to identify the molecular pathways and putative targets regulated by miR-218.The luciferase reporter assay was applied to identify the actual binding site of miR-218 in target gene. The expression of LASP1 was investigated by immunohistochemistry.RESULTS: The expression levels of miR-218 were significantly downregulated in PCa tissues and PCa cell lines. Restoration of miR-218 in PCa cell lines revealed that miR-218 significantly inhibited cancer cell migration and invasion.Gene expression studies and luciferase reporter assays showed that LASP1 was directly regulated by miR-218. Silencing LASP1 resulted in significant inhibition of cell migration and invasion in PCa cells.The expression of LASP1 was upregulated in cancer tissues as demonstrated byimmunohistochemical staining.CONCLUSIONS: The miR-218 was frequently reduced in PCa clinical specimens and appeared to function as a tumor suppressor through regulation of oncogenic LASP1. The identification of novel molecularpathways and targets regulated by the tumor suppressive miRNAs may lead to a better understanding of PCa progression and metastasis, and the development of new therapeutic strategies to treat this disease. Citation Format: Rika Nishikawa, Yusuke Goto, Takashi Kinoshita, Shinichi Sakamoto, Takeshi Chiyomaru, Hideki Enokida, Satoko Kojima, Masayuki Nakagawa, Yukio Naya, Tomohiko Ichikawa, Naohiko Seki. Tumor suppressive microRNA-218 inhibits cancer cell migration and invasion via targeting LASP1 in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4371. doi:10.1158/1538-7445.AM2014-4371

Abstract 4350: Tumor-suppressive microRNA-29a inhibits cancer cell migration and invasion via targeting HSP47 in cervical squamous cell carcinoma

Abstract BACKGROUNDS: Cervical cancer is the second most common cause of cancer death in women worldwide, and approximately 500,000 new cases of cervical cancer are diagnosed each year, with 280,000 deaths. Cervical squamous cell carcinoma (SCC) is the most frequent type of cervical cancer and the most important risk factor for cervical-SCC is persistent human papilloma virus (HPV) infection. However, the molecular mechanisms of cervical SCC initiation, development, and metastasis have not yet been fully elucidated. Our recent studies of microRNA (miRNA) expression signatures have indicated that miRNA-29a (miR-29a) was significantly downregulated in several types of human cancers, suggesting that miR-29a may be a putative tumor-suppressive miRNA in human cancers. The aim of this study was to investigate the functional significance of miR-29a in cervical squamous cell carcinoma (SCC) and to identify novel miR-29a-regulated cancer pathways and target genes involved in cervical SCC oncogenesis and metastasis. METHODS: The expression levels of miR-29a were validated using real-time RT-PCR using cervical SCC clinical specimens and cell lines (CaSKi, HeLa, ME180, and YUMOTO). Cell proliferation, migration and invasion assays were performed to investigate the functional significance of miR-29a and its target gene, heat-shock protein 47 (HSP47) in cell lines. Cells were transfected with mature miR-29a or si-HSP47 by reverse transfection methods. Genome-wide gene expression data and in silico analysis were used to identify the molecular pathways and putative targets regulated by miR-29a. The luciferase reporter assay was applied to identify the actual binding site of miR-29a in target gene. The expression of HSP47 was investigated by immunohistochemistry. RESULTS: Restoration of miR-29a in cervical cancer cell lines revealed that this miRNA significantly inhibited cancer cell migration and invasion. Gene expression data and in silico analysis demonstrated that HSP47 was a potential target of miR-29a regulation. Luciferase reporter assays showed that miR-29a directly regulated HSP47. Moreover, silencing of the HSP47 gene significantly inhibited cell migration and invasion in cancer cells, and the expression of HSP47 was upregulated in cancer tissues and cervical intraepithelial neoplasia (CIN), as demonstrated by immunostaining. CONCLUSIONS: Downregulation of miR-29a was a frequent event in cervical SCC, and miR-29a acted as a tumor suppressor by directly targeting HSP47. Recognition of tumor-suppressive miRNA-regulated molecular targets provides new insights into the potential mechanisms of cervical SCC oncogenesis and metastasis and suggests novel therapeutic strategies for treatment of this disease. Citation Format: Noriko Yamamoto, Takashi Kinoshita, Nijiro Nohata, Akira Mitsuhashi, Hirokazu Usui, Hirofumi Yoshino, Toshihiko Itesako, Hideki Enokida, Masayuki Nakagawa, Makio Shozu, Naohiko Seki. Tumor-suppressive microRNA-29a inhibits cancer cell migration and invasion via targeting HSP47 in cervical squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4350. doi:10.1158/1538-7445.AM2014-4350

Abstract 4352: Tumor suppressive microRNAs (miR-29s/miR-218) regulate laminin-integrin signaling in head and neck squamous cell carcinoma

Abstract BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. In spite of advances in the therapy, the overall five-year survival rate for patients with HNSCC is around 40%. Distant metastasis after conventional therapy appears to be a major contributing factor for the restricted survival of HNSCC patients. Understanding the molecular pathways of HNSCC metastasis would help to improve diagnosis, approaches to therapy and prevention of the disease. Our recent study of microRNA (miRNA) expression signature of HNSCC has revealed that microRNA-29s (miR-29s) and microRNA-218 (miR-218) were significantly downregulated in cancer tissues, suggesting that these miRNAs are candidate of tumor suppressors. The aim of the study was to investigate the functional significance of miR-29s and miR-218 in HNSCC cells and to identify novel metastatic pathways regulated by these miRNAs. METHODS: Cell proliferation, migration and invasion assays were performed to investigate the functional significance of miR-29s (miR-29a/b/c) and miR-218 and these target genes in HNSCC cell lines (FaDu and SAS). Cells were transfected with mature miRNAs or siRNAs by reverse transfection methods. Genome-wide gene expression data and in silico analysis were used to identify the molecular pathways and putative targets regulated by miR-29s or miR-218. The luciferase reporter assays were used to identify the actual binding sites of target genes regulated by these miRNAs. RESULTS: Restoration of miR-29s or miR-218 in HNSCC cells revealed that these miRNAs significantly inhibited cancer cell migration and invasion. Genome-wide gene expression data and in silico analysis showed that focal adhesion pathway was a promising candidate of miR-29s and miR-218 target pathway. Interestingly, interaction between laminin-332 (LAMA3, LAMB3 and LAMC2) and α6β4 integrin (ITGA6 and ITGB4) triggers a number of signalling cascades, promoting both cell migration and cancer cell survival. Luciferase reporter assays showed that LAMC2 and ITGA6 were directly regulated by miR-29s. Also, LAMB3 was regulated by miR-218. Silencing of LAMB3, LAMC2 and ITGA6 genes significantly inhibited cell migration and invasion in cancer cells. CONCLUSIONS: Downregulation of miR-29s and miR-218 were frequent events in HNSCC. These miRNAs acted as tumour suppressors and directly targeted laminin-integrin signalling. Recognition of tumour suppressive miRNA-mediated cancer pathways provides new insights into the potential mechanisms of HNSCC oncogenesis and metastasis and suggests novel therapeutic strategies for the disease. Citation Format: Takashi Kinoshita, Nijiro Nohata, Toyoyuki Hanazawa, Naoko Kikkawa, Noriko Yamamoto, Hirofumi Yoshino, Toshihiko Itesako, Hideki Enokida, Masayuki Nakagawa, Yoshitaka Okamoto, Naohiko Seki. Tumor suppressive microRNAs (miR-29s/miR-218) regulate laminin-integrin signaling in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4352. doi:10.1158/1538-7445.AM2014-4352

High-strength photoresponsive hydrogels enable surface-mediated gene delivery and light-induced reversible cell adhesion/detachment.

In the present study, high-strength photoresponsive hydrogels were prepared by the photoinitiated copolymerization of acrylamide (AAm, hydrophilic hydrogen bonding monomer), 2-vinyl-4,6-diamino-1,3,5-triazine (VDT, hydrophobic hydrogen bonding monomer), and spiropyran-containing monomer (SPAA) in the presence of cross-linker poly(ethylene glycol) diacrylate (PEG575DA, Mn = 575). The double hydrogen bondings from AAm-AAm and diaminotriazine-diaminotriazine contributed to the considerable enhancement in tensile and compressive properties of the hydrogels, which showed an excellent ability to resist a variety of external forces. Fifteen minutes of UV (365 nm) irradiation led to the detachment of adhered cells due to the increased surface hydrophilicity caused by the isomerization of spiropyran moieties. Furthermore, repeated attachment/detachment of cells was realized by the alternate illumination of visible and UV light. Reverse gene transfection was carried out successfully by anchoring the PVDT/pDNA complex nanoparticles on the gel surface through hydrogen bonding between diaminotriazine motifs prior to cell seeding. Importantly, fibronectin (FN) modification combined with supplementing PVDT/pDNA complex nanoparticles after the first cycle of reverse gene transfection, so-called sandwich gene transfection, further increased the gene transfection level. A short time of UV light exposure could result in the nonharmful detachment of gene-modified cells from the gel surface. This high-strength photosensitive hydrogel holds potential as a reusable soft-wet platform for cell harvesting as well as gene transfection operation at higher efficiency.

183 Novel OPG Protein Interactions Regulate Survival, Proliferation And Pah-associated Gene Expression in Pulmonary Arterial Smooth Muscle Cells

IntroductionPulmonary arterial hypertension (PAH) is a devastating disease with a high mortality and prognosis worse than many cancers. Pathologically, PAH is characterised by progressive arteriole remodelling driven by pulmonary artery...

544 Comprehensive Profiling of Plasma MicroRNAs Reveals Potential Biomarkers for Barrett's Esophagus and Esophageal Adenocarcinoma

Aim: To quantitatively assess complex cancer cell phenotypes after gene inhibition by RNAi interference (RNAi), and assess the effect of these distinct morphologies on cell viability and cell motility using semi-automated image-based high-throughput screening. Method: GOhTRT cells were seeded and treated with the siRNA Human Druggable Genome Library (Dharmacon) by reverse transfection. Cells immunostained for DNA, tubulin and actin were imaged with the InCell Analyzer 1000 and processed using the InCell Analyzer software, CellHTS2 and RNAither. Statistical z-score analysis was performed on the combined A-T metric (F-actin area-α-tubulin area). The effect of RNAi knockdown on cell viability and cell motility were assessed using MTT cell proliferation assay and scratch wound assay. Results:127 high confidence hits (Z-factor>2) was refined to six genes (RRM2, ITGB8, GPS1, SPRY1, NOL1, MYO9B) on the basis of distinct morphologies, reproducible metrics, and functional pathway analysis. In siRRM2 cells nuclear displacement (ND) and A-T area was 1.598±0.076; p <0.0001 and 285.70±35.48; p<0.05, respectively. siGPS1 cells had an ND and A-T area of 0.845±0.036; p =0.0369 and 266.201±25.629; p =0.0326, respectively. Silencing of GPS1, MYO9B and SPRY1 increased the rate of migration in a scratch wound assay, with 86.98%±3.097%, 75.78%±5.454% and 72.97%±5.463% (p =0.0022) respectively. No significant difference in cell viability existed for siGPS1 (0.9037 ± 0.06575; p = 0.1905) and siSPRY1 (0.9088 ± 0.09849; p =0.2985), suggesting that wound closure is by virtue of migratory signalling and not proliferation. Cell viability was decreased considerably in siRRM2 cells (0.2492±0.02798; p <0.0001) and siMYO9B (0.4048±0.04663; p <0.0001) in comparison to siNT cells (1.046±0.07712). Discussion:In summary, this approach successfully identified genes regulating oesophageal cancer cell cytoskeletal remodelling and metastasis using in-vitro assays, some of which are already associated with metastasis in literature and database searches. Further mechanistic studies and gene pathway analysis of candidate genes will provide novel therapeutic targets which can be utilised to block the spread of cancer in oesophageal adenocarcinoma patients.

546 Genome-Wide Gene-Environment Interaction Analysis of Aspirin and Non-Steroidal Anti-Inflammatory Drug Use and Colorectal Cancer Identifies Loci At Chromosomes 12p12.3 and 15q25.2

Aim: To quantitatively assess complex cancer cell phenotypes after gene inhibition by RNAi interference (RNAi), and assess the effect of these distinct morphologies on cell viability and cell motility using semi-automated image-based high-throughput screening. Method: GOhTRT cells were seeded and treated with the siRNA Human Druggable Genome Library (Dharmacon) by reverse transfection. Cells immunostained for DNA, tubulin and actin were imaged with the InCell Analyzer 1000 and processed using the InCell Analyzer software, CellHTS2 and RNAither. Statistical z-score analysis was performed on the combined A-T metric (F-actin area-α-tubulin area). The effect of RNAi knockdown on cell viability and cell motility were assessed using MTT cell proliferation assay and scratch wound assay. Results:127 high confidence hits (Z-factor>2) was refined to six genes (RRM2, ITGB8, GPS1, SPRY1, NOL1, MYO9B) on the basis of distinct morphologies, reproducible metrics, and functional pathway analysis. In siRRM2 cells nuclear displacement (ND) and A-T area was 1.598±0.076; p <0.0001 and 285.70±35.48; p<0.05, respectively. siGPS1 cells had an ND and A-T area of 0.845±0.036; p =0.0369 and 266.201±25.629; p =0.0326, respectively. Silencing of GPS1, MYO9B and SPRY1 increased the rate of migration in a scratch wound assay, with 86.98%±3.097%, 75.78%±5.454% and 72.97%±5.463% (p =0.0022) respectively. No significant difference in cell viability existed for siGPS1 (0.9037 ± 0.06575; p = 0.1905) and siSPRY1 (0.9088 ± 0.09849; p =0.2985), suggesting that wound closure is by virtue of migratory signalling and not proliferation. Cell viability was decreased considerably in siRRM2 cells (0.2492±0.02798; p <0.0001) and siMYO9B (0.4048±0.04663; p <0.0001) in comparison to siNT cells (1.046±0.07712). Discussion:In summary, this approach successfully identified genes regulating oesophageal cancer cell cytoskeletal remodelling and metastasis using in-vitro assays, some of which are already associated with metastasis in literature and database searches. Further mechanistic studies and gene pathway analysis of candidate genes will provide novel therapeutic targets which can be utilised to block the spread of cancer in oesophageal adenocarcinoma patients.

Heat Shock Protein 70 Modulates Influenza A Virus Polymerase Activity

The role of heat shock protein 70 (Hsp70) in virus replication has been discussed for many viruses. The known suppressive role of Hsp70 in influenza virus replication is based on studies conducted in cells with various Hsp70 expression levels. In this study, we determined the role of Hsp70 in influenza virus replication in HeLa and HEK293T cells, which express Hsp70 constitutively. Co-immunoprecipitation and immunofluorescence studies revealed that Hsp70 interacted with PB2 or PB1 monomers and PB2/PB1 heterodimer but not with the PB1/PA heterodimer or PB2/PB1/PA heterotrimer and translocated into the nucleus with PB2 monomers or PB2/PB1 heterodimers. Knocking down Hsp70 resulted in reduced virus transcription and replication activities. Reporter gene assay, immunofluorescence assay, and Western blot analysis of nuclear and cytoplasmic fractions from infected cells demonstrated that the increase in viral polymerase activity during the heat shock phase was accompanied with an increase in Hsp70 and viral polymerases levels in the nuclei, where influenza virus replication takes place, whereas a reduction in viral polymerase activity was accompanied with an increase in cytoplasmic relocation of Hsp70 along with viral polymerases. Moreover, significantly higher levels of viral genomic RNA (vRNA) were observed during the heat shock phase than during the recovery phase. Overall, for the first time, these findings suggest that Hsp70 may act as a chaperone for influenza virus polymerase, and the modulatory effect of Hsp70 appears to be a sequel of shuttling of Hsp70 between nuclear and cytoplasmic compartments.

Gene-modified cell detachment on photoresponsive hydrogels strengthened through hydrogen bonding

Photoresponsive hydrogels are potentially useful as drug delivery and cell culture media, but there has been no report on manipulation of cell attachment/detachment and gene transfection simultaneously on the surface of this single gel. In the present study, strong light sensitive hydrogels were prepared mechanically by photoinitiated copolymerization of spiropyran-containing monomer, 2-vinyl-4,6-diamino-1,3,5-triazine, hydrogen bonding monomer, oligo(ethylene glycol) methacrylate and polyethylene glycol diacrylate (PEGDA, Mn=575). The multiple hydrogen bondings of diaminotriazine residues could contribute to the increase in compressive strengths of the photosensitive hydrogels up to 5.1MPa. UV (365nm) irradiation led to detachment of adhered cells as a result of the increased surface hydrophilicity caused by a switch from hydrophobic spiropyran to hydrophilic merocyanine form. Furthermore, selective detachment of cells could also be achieved by UV light illumination on the specified gel surface. Hydrogen bonding between diaminotriazines were shown to tightly anchor the PVDT/pDNA complex particles on the gel surface, where reverse gene transfection was achieved. Following up with UV irradiation triggered the unharmful detachment of gene-modified cells from the gel surface. It is envisioned that this photosensitive hydrogel holds potential as a versatile platform for operating gene delivery and controlled harvest of desired cells for tissue engineering.

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson's disease. Acute activation of Gαi/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of Gαi/o-linked receptors including D2 dopamine and μ opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gbγ signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384 well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e. CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (>20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

A High-Throughput Assay to Identify Modifiers of Premature Chromosome Condensation

Premature chromosome condensation (PCC) is a consequence of early mitotic entry, where mitosis begins before completion of DNA replication. Previously we have identified mutations in MCPH1, a DNA damage response and potential tumor suppressor gene, as a cause of primary microcephaly and PCC. Here we describe a high-throughput assay to identify modifiers of PCC. Reverse transfection of control siRNA followed by a forward transfection of MCPH1 small interfering RNA (siRNA) was performed to induce PCC. Condensin II subunits CAPG2 and CAPH2 were validated as PCC modifiers and therefore positive controls. Cell nuclei were detected by DAPI staining using an Operetta imaging system. PCC and nuclei number were determined using Columbus analysis software. Two batches of nine plates were used to determine assay efficacy. Each plate contained four negative (nontargeting) and eight positive control siRNAs. Mean % PCC was 12.35% (n = 72) for negative controls and 4.25% (n = 144) for positive controls. Overall false-positive and false-negative rates were 0% (n = 72) and 2.1% (n = 144), respectively. This assay is currently being used to screen a human druggable genome siRNA library to identify novel therapeutic targets for cancer treatment. The assay can also be used to identify novel compounds and genes that induce PCC.

Reverse transfection in microchamber arrays for cell migration assays

A microdevice was designed to prevent cross-contamination of cells and reagents among reverse transfection (RTF) spots by using nanoliter-scale culture chambers. The forces generated by surface tension were used to seed cells into all the culture chambers simultaneously and separate them for following migration assays. Using the microdevice, bladder carcinoma cells were reverse-transfected and a migration-related gene was detected by tracing transfected cells. This approach may provide a platform for robust RTF assay systems.

Cell-based high-throughput odorant screening system through visualization on a microwell array

The development of a cell-based high-throughput screening system has attracted much attention from researchers who study drug screening mechanisms and characterization of G-protein coupled receptors (GPCRs). Although olfactory receptors (ORs) constitute the largest group of GPCRs that play a critical role recognizing and discriminating odorants, only a few ORs have been characterized, and most remain orphan. The conventional cell-based assay system for characterizing GPCRs, including ORs, is very laborious, time consuming, and requires an expensive assay system. In this study, we developed a simple, low-cost miniaturized odorant screening method by combining Micro-Electro-Mechanical system (MEMs) technique and visualization technique for detecting an odorant response. We fabricated PEG microwell from a photocrosslinkable polyethylene glycol diacrylate (PEGDA) solution and applied it to cell culture and a reverse transfection platform for cell-based high-throughput screening. For the first time, the olfactory receptors were expressed on the microwell platform using reverse transfection technique. The various olfactory receptors can be expressed simultaneously using this technique and the microwell spotted with olfactory receptor genes can be used as a high-throughput screening platform. The odorant response was detected via fluorescence analysis on the microwell using a cAMP response element (CRE) reporter assay. We tested this platform using four de-orphaned ORs. This new cell-based screening method not only reduced numerous time-consuming steps but also allowed for simple, efficient, and quantitative screening and patterning of large numbers of GPCRs including ORs, which can help to visualize the OR response to odorants on a microwell.

Facile and Multiple Replication of Superhydrophilic–Superhydrophobic Patterns Using Adhesive Tape

Surfaces patterned with both hydrophilic and hydrophobic regions are useful in a variety of applications. For example, they can be used as surface tension-confined microchannels, in paper-based microfluidics, or for patterning cells. To create a new patterned substrate, usually the entire experimental procedure must be repeated, which can be time-consuming and laborious. In this paper, we present a simple and fast method that allows the transfer of superhydrophilic-superhydrophobic micropatterns in porous polymer films onto adhesive tape. Replicating patterns using adhesive tape is economical, as the fabrication of one patterned substrate can be used to create multiple copies of the micropatterns, which can then be used for several different experiments. We demonstrate that at least twelve consecutive copies can be made from 125 μm-thick patterned polymer films. Since the polymer film is transferred to adhesive tape, which is flexible, the copies can be used on curved surfaces and they can also be cut into different shapes and sizes. We also demonstrate an application of the replicated patterned polymer surfaces as a substrate for reverse cell transfection experiments.

Abciximab-Krueppel-like factor 4-plasmid-titanium dioxide coated coronary stent for inhibition of neointimal hyperplasia

Purpose: We developed dual system in delivery of gene and drug for coronary stent. For local gene delivery, denatured polymer coating was used. For drug delivery, titanium dioxide (TiO2) thin film which was non-polymer and had a high blood compatibility was used. In this study, we hypothesized whether dual delivery system of gene and drug for coronary stent was useful in preventing in-stent restenosis (ISR). Methods and results: The coating and modification of TiO2 thin film were manipulated by plasma enhanced chemical vapor deposition (PECVD) method. In vitro study, TiO2-Drug (Abciximab) coating stainless steel (SS) plate catch a larger amount of gWIZ-beta gal (beta-gal) plasmid compared with TiO2-only coating SS plate (2.70±0.32 vs. 7.17±0.38 mg/cm2, p<0.05). Also, the binding plasmid (Krueppel-Like Factor 4, KLF4) was effectively transfected into 293 cells after detachment using plasmid elution buffer. The reverse transfection using direct attachment of pig coronary artery smooth muscles showed the X-gal stain positive cells after 5 days. For analysis of in vivo gene delivery, we used animal models of rat abdominal skin and pig coronary artery. At 5 days after implantation of TiO2-Drug-beta-gal plasmid SS plate in rat abdominal skin, the expression of the beta-gal plasmid was observed. At 5 days after implantation of TiO2-Drug-beta-gal plasmid binding coronary stent in balloon-injured porcine coronary artery restenosis model, X-gal stained linear area was observed. To analyze the inhibitory effect of ISR, we used both TiO2-Abciximab-KLF4-plasmid-cobalt chromium (TAK-CC) and TiO2-only coating CC (T-CC) stent. Four weeks after implantation in porcine coronary artery, in histomorphometric analysis, TAK-CC stent group decreased ISR compared with T-CC group (TAK-CC group: 26.38±10.79% vs. T-CC group: 48.59±20.17%, n=6 in each, p<0.05). Also, both TAK-CC and T-CC stent group did not show the fibrin deposition at Castars' stain and accelerated re-endothelialization indicated by detectable anti-CD31 positive stained cells compared with commercial available paclitaxel-eluting stent. Conclusions: Our study implicate that this novel new system could be useful for coronary stent and other biomedical devices through dual delivery of gene and drug.

Cyclodextrin-cross-linked diaminotriazine-based hydrogen bonding strengthened hydrogels for drug and reverse gene delivery

A hydrogen bonding strengthened hydrogel was prepared by radical copolymerization of poly(ethylene glycol) methacrylated β-cyclodextrin (PEG-β-CD) and 2-vinyl-4,6-diamino-1,3,5-triazine (VDT) monomer. PEG-β-CD served not only as a cross-linker, but also as a built-in solubilizing agent of the hydrophobic drug in the gel. Increasing VDT content resulted in a notable enhancement in the mechanical strengths of hydrogels whose equilibrium water contents could be modulated from 75% to 85% by varying the ratio of PEG-β-CD cross-linker. It was shown that copolymerizing more PEG-β-CDs could load higher amount of ibuprofen (IBU) in the gels and contribute to a slower release rate of IBU. Plasmid DNA could be anchored onto the surface of hydrogels due to the hydrogen bonding between the base pairs and diaminotriazine, thereby mediating efficient reverse gene transfection of luciferase gene in COS-7 cells cultured on the gel surface. The cytocompatible PEG-β-CD-cross-linked PVDT hydrogels with multifunction of drug and gene delivery hold a potential as tissue engineering scaffold.

Abstract B40: X-MANTM isogenic disease models: Precision tools for synthetic lethality screening

Abstract Cancer is a genetic disease, and as such, genetic variation is almost exclusively responsible for onset and progression, and critically is also a key component in an individual's response to therapeutics. Understanding the complex of genetics of cancer and identifying the key mutations driving phenotype requires patient-relevant disease models. Horizon Discovery's expertise in translational genomics has led to the development of GENESIS™ a proprietary genome-editing platform based on the unique ability of rAAV to stimulate homologous recombination. By exploiting a cell's natural means of repairing its own DNA in an error-free manner, rAAV GENESIS™ enables the precise insertion, deletion or substitution of a desired DNA sequence into any endogenous gene loci in mammalian cells. Horizon has generated over 500 X-MAN™ (gene-X mutant and normal) isogenic cell lines, the world's first source of genetically-defined and patient-relevant human cellular models. We are currently using multiple panels of these disease models in combination with high-throughput RNAi screens to create a large scale synthetic lethality target identification platform. Our platform consists of lines that are identical except for the targeted modification, enabling us to identify gene target hits that have tumor selective properties. Here we have selected isogenically matched HCT116 parental, p53 null (-/-) and p53 mutant (R248W/+) lines; by use of reverse transfection methodologies we achieved &amp;gt;80% knockdown efficiency for a library of over 2,200 druggable targets. This approach yielded targets that selectively induce lethality in the presence of a p53 mutation for both the null (-/-) and mutant lines (R248W/+), interestingly two of the top hits inducing synthetic lethality in the p53 null lines are p21 and the p21 binding partner CDKN1A. The full power of these experiments comes from comparisons of the isogenically matched lines. Our results demonstrate that the p53 null lines (-/-) have a marked resistance to siRNA mediated synthetic lethality when compared to the mutant (R248W/+) lines, hit rates being 0.6% and 13% respectively. The number of p53 R248W/+ mutant hits are also of great interest. It is well reported that p53 transgenic mice rapidly succumb to certain types of cancers not commonly observed in p53 null mice (Song et. al. Nat Cell Biol. 2007). It is thought this mutation promotes tumorigenesis by a novel mechanism involving active disruption of critical DNA damage-response pathways. This mechanism of action clearly renders these cells more susceptible to therapeutic intervention. Overall these results highlight the challenge of identifying therapeutics that are able to target p53 null tumors. The mutant (R248W/+) hits, however may offer a therapeutic opportunity, especially when considering that mutations at this locus are the most commonly occurring p53 mutations reported in human tumors. Citation Format: Jo Francis, Jessica Hunt, Kyla Grimshaw, Agustin Chicas, Lihua Yu, Ping Zhu, Markus Warmuth, Chris Torrance. X-MANTM isogenic disease models: Precision tools for synthetic lethality screening. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B40.