A cell spot microarray method for high-throughput biology

High-throughput screening of cellular effects of RNA interference (RNAi) libraries is now being increasingly applied to explore the role of genes in specific cell biological processes and disease states. However, the technology is still limited to specialty laboratories, due to the requirements for robotic infrastructure, access to expensive reagent libraries, expertise in high-throughput screening assay development, standardization, data analysis and applications. In the future, alternative screening platforms will be required to expand functional large-scale experiments to include more RNAi constructs, allow combinatorial loss-of-function analyses (e.g. genegene or gene-drug interaction), gain-of-function screens, multi-parametric phenotypic readouts or comparative analysis of many different cell types. Such comprehensive perturbation of gene networks in cells will require a major increase in the flexibility of the screening platforms, throughput and reduction of costs. As an alternative for the conventional multi-well based high-throughput screening -platforms, here the development of a novel cell spot microarray method for production of high density siRNA reverse transfection arrays is described. The cell spot microarray platform is distinguished from the majority of other transfection cell microarray techniques by the spatially confined array layout that allow highly parallel screening of large-scale RNAi reagent libraries with assays otherwise difficult or not applicable to high-throughput screening. This study depicts the development of the cell spot microarray method along with biological application examples of high-content immunofluorescence and phenotype based cancer cell biological analyses focusing on the regulation of prostate cancer cell growth, maintenance of genomic integrity in breast cancer cells, and functional analysis of integrin protein-protein interactions in situ.RNA-interferenssin (RNAi) käyttö geenituotteiden toimintojen tutkimuksessa on vuosikymmenessä kehittynyt solubiologisen tutkimuksen merkittävimpien teknologioiden joukkoon. RNAi-menetelmät ovat mahdollistaneet myös eri solubiologisten signaalivälitysreittien kartoittamisen koko perimänlaajuisten geenitoimintojen suurtehoseulonnan avulla. Teknologian nopeasta kehityksestä ja laajamittaisesta hyödyntämisestä huolimatta, RNAi-menetelmien käyttö suurtehoseulontaan rajoittuu edelleen erikoislaboratorioihin huomattavien laite-, reagenssi- sekä tietotaitovaatimusten johdosta. Lisäksi yleisimmin käytetyt kuoppalevypohjaiset seulonta-menetelmät rajoittavat seulontatutkimusten laajuutta, eri analyysimenetelmien käyttöä sekä useiden mallisolulinjojen rinnakkaista seulontaa suuren reagenssikulutuksen takia. RNAi-suurtehoseulonnan kustannuksien ja laiteriippuvuuden alentamiseksi sekä monipuolisuuden lisäämiseksi tulevaisuudessa tarvitaan uusia vaihtoehtoisia seulontamenetelmiä. Tämän väitöskirjatyön lähtökohtana oli uuden kuoppalevyillä tehtäviä seulontamenetelmiä edullisemman ja monipuolisemman RNAi-suurtehoseulontamenetelmän kehittäminen. Tutkimuksessa kehitettiin solujen siRNA käänteistransfektioon perustuva solumikrosirumenetelmä, joka tarkasti rajattujen siRNA näytepisteiden avulla mahdollistaa jopa koko ihmisen perimänlaajuisten siRNA näytekirjastojen seulonnan yksittäisellä mittasirulla. Menetelmän suuri näytetiheys sekä sirujen pieni pinta-ala mahdollistavat myös erikoismittamenetelmien käytön suurtehoseulonnassa, jotka teknisistä tai kustannussyistä eivät sovellu käytettäväksi suurtehoseulontaan kuoppalevyillä. Väitöskirjan ensimmäinen osatyö kuvaa menetelmän kehittämistyön. Väitöskirjan toisessa, kolmannessa ja neljännessä osatyössä menetelmää on hyödynnetty syöpäbiologisissa seulontatutkimuksissa selvitettäessä geenejä jotka vaikuttavat eturauhassyöpäsolujen kasvuun, rintasyöpäsolujen solunjakautumiseen sekä integriini tarttumisreseptorien aktiivisuuden säätelyyn.Siirretty Doriast

The gene expression landscape of breast cancer is shaped by tumor protein p53 status and epithelial-mesenchymal transition

Introduction: Gene expression data derived from clinical cancer specimens provide an opportunity to characterize cancer-specific transcriptional programs. Here, we present an analysis delineating a correlation-based gene expression landscape of breast cancer that identifies modules with strong associations to breast cancer-specific and general tumor biology. Methods: Modules of highly connected genes were extracted from a gene co-expression network that was constructed based on Pearson correlation, and module activities were then calculated using a pathway activity score. Functional annotations of modules were experimentally validated with an siRNA cell spot microarray system using the KPL-4 breast cancer cell line, and by using gene expression data from functional studies. Modules were derived using gene expression data representing 1,608 breast cancer samples and validated in data sets representing 971 independent breast cancer samples as well as 1,231 samples from other cancer forms. Results: The initial co-expression network analysis resulted in the characterization of eight tightly regulated gene modules. Cell cycle genes were divided into two transcriptional programs, and experimental validation using an siRNA screen showed different functional roles for these programs during proliferation. The division of the two programs was found to act as a marker for tumor protein p53 (TP53) gene status in luminal breast cancer, with the two programs being separated only in luminal tumors with functional p53 (encoded by TP53). Moreover, a module containing fibroblast and stroma-related genes was highly expressed in fibroblasts, but was also up-regulated by overexpression of epithelial-mesenchymal transition factors such as transforming growth factor beta 1 (TGF-beta1) and Snail in immortalized human mammary epithelial cells. Strikingly, the stroma transcriptional program related to less malignant tumors for luminal disease and aggressive lymph node positive disease among basal-like tumors. Conclusions: We have derived a robust gene expression landscape of breast cancer that reflects known subtypes as well as heterogeneity within these subtypes. By applying the modules to TP53-mutated samples we shed light on the biological consequences of non-functional p53 in otherwise low-proliferating luminal breast cancer. Furthermore, as in the case of the stroma module, we show that the biological and clinical interpretation of a set of co-regulated genes is subtype-dependent

Ex vivo drug screening informed targeted therapy for metastatic parotid squamous cell carcinoma

The purpose of ex vivo drug screening in the context of precision oncology is to serve as a functional diagnostic method for therapy efficacy modeling directly on patient-derived tumor cells. Here, we report a case study using integrated multiomics ex vivo drug screening approach to assess therapy efficacy in a rare metastatic squamous cell carcinoma of the parotid gland. Tumor cells isolated from lymph node metastasis and distal subcutaneous metastasis were used for imaging-based single-cell resolution drug screening and reverse-phase protein array-based drug screening assays to inform the treatment strategy after standard therapeutic options had been exhausted. The drug targets discovered on the basis of the ex vivo measured drug efficacy were validated with histopathology, genomic profiling, and in vitro cell biology methods, and targeted treatments with durable clinical responses were achieved. These results demonstrate the use of serial ex vivo drug screening to inform adjuvant therapy options prior to and during treatment and highlight HER2 as a potential therapy target also in metastatic squamous cell carcinoma of the salivary glands

Integrin trafficking regulated by Rab21 is necessary for cytokinesis

Adherent cells undergo remarkable changes in shape during cell division. However, the functional interplay between cell adhesion turnover and the mitotic machinery is poorly understood. The endo/exocytic trafficking of integrins is regulated by the small GTPase Rab21, which associates with several integrin alpha subunits. Here, we show that targeted trafficking of integrins to and from the cleavage furrow is required for successful cytokinesis, and that this is regulated by Rab21. Rab21 activity, integrin-Rab21 association, and integrin endocytosis are all necessary for normal cytokinesis, which becomes impaired when integrin-mediated adhesion at the cleavage furrow fails. We also describe a chromosomal deletion and loss of Rab21 gene expression in human cancer, which leads to the accumulation of multinucleate cells. Importantly, reintroduction of Rab21 rescued this phenotype. In conclusion, Rab21-regulated integrin trafficking is essential for normal cell division, and its defects may contribute to multinucleation and genomic instability, which are hallmarks of cancer.Adherent cells undergo remarkable changes in shape during cell division. However, the functional interplay between cell adhesion turnover and the mitotic machinery is poorly understood. The endo/exocytic trafficking of integrins is regulated by the small GTPase Rab21, which associates with several integrin alpha subunits. Here, we show that targeted trafficking of integrins to and from the cleavage furrow is required for successful cytokinesis, and that this is regulated by Rab21. Rab21 activity, integrin-Rab21 association, and integrin endocytosis are all necessary for normal cytokinesis, which becomes impaired when integrin-mediated adhesion at the cleavage furrow fails. We also describe a chromosomal deletion and loss of Rab21 gene expression in human cancer, which leads to the accumulation of multinucleate cells. Importantly, reintroduction of Rab21 rescued this phenotype. In conclusion, Rab21-regulated integrin trafficking is essential for normal cell division, and its defects may contribute to multinucleation and genomic instability, which are hallmarks of cancer.Adherent cells undergo remarkable changes in shape during cell division. However, the functional interplay between cell adhesion turnover and the mitotic machinery is poorly understood. The endo/exocytic trafficking of integrins is regulated by the small GTPase Rab21, which associates with several integrin alpha subunits. Here, we show that targeted trafficking of integrins to and from the cleavage furrow is required for successful cytokinesis, and that this is regulated by Rab21. Rab21 activity, integrin-Rab21 association, and integrin endocytosis are all necessary for normal cytokinesis, which becomes impaired when integrin-mediated adhesion at the cleavage furrow fails. We also describe a chromosomal deletion and loss of Rab21 gene expression in human cancer, which leads to the accumulation of multinucleate cells. Importantly, reintroduction of Rab21 rescued this phenotype. In conclusion, Rab21-regulated integrin trafficking is essential for normal cell division, and its defects may contribute to multinucleation and genomic instability, which are hallmarks of cancer.Peer reviewe

Integrative functional genomics analysis of sustained polyploidy phenotypes in breast cancer cells identifies an oncogenic profile for GINS2

Aneuploidy is among the most obvious differences between normal and cancer cells. However, mechanisms contributing to development and maintenance of aneuploid cell growth are diverse and incompletely understood. Functional genomics analyses have shown that aneuploidy in cancer cells is correlated with diffuse gene expression signatures and that aneuploidy can arise by a variety of mechanisms, including cytokinesis failures, DNA endoreplication and possibly through polyploid intermediate states. Here, we used a novel cell spot microarray technique to identify genes with a loss-of-function effect inducing polyploidy and/or allowing maintenance of polyploid cell growth of breast cancer cells. Integrative genomics profiling of candidate genes highlighted GINS2 as a potential oncogene frequently overexpressed in clinical breast cancers as well as in several other cancer types. Multivariate analysis indicated GINS2 to be an independent prognostic factor for breast cancer outcome (p = 0.001). Suppression of GINS2 expression effectively inhibited breast cancer cell growth and induced polyploidy. In addition, protein level detection of nuclear GINS2 accurately distinguished actively proliferating cancer cells suggesting potential use as an operational biomarker.Peer reviewe

Cisplatin overcomes radiotherapy resistance in OCT4-expressing head and neck squamous cell carcinoma

Objectives: Cisplatin is combined with radiotherapy for advanced head and neck squamous cell carcinoma (HNSCC). While providing a beneficial effect on survival, it also causes side effects and thus is an important target when considering treatment de-escalation. Currently, there are no biomarkers to predict its patientselective therapeutic utility. In this study, we examined the role of the stem cell factor OCT4 as a potential biomarker to help clinicians stratify HNSCC patients between radiotherapy and chemoradiotherapy. Materials and methods: OCT4 immunohistochemical staining of a population-validated tissue microarray (PV-TMA) (n = 166) representative of a standard HNSCC patients was carried out, and 5-year survival was analyzed. The results were validated using ex vivo drug sensitivity analysis of HNSCC tumor samples, and further crossvalidated in independent oropharyngeal (n = 118), nasopharyngeal (n = 170), and vulvar carcinoma (n = 95) clinical datasets. In vitro, genetically modified, patient-derived HNSCC cells were used. Results: OCT4 expression in HNSCC tumors was associated with radioresistance. However, combination therapy with cisplatin was found to overcome this radioresistance in OCT4-expressing HNSCC tumors. The results were validated by using several independent patient cohorts. Furthermore, CRISPRa-based OCT4 overexpression in the HNSCC cell line resulted in apoptosis resistance, and cisplatin was found to downregulate OCT4 protein expression in vitro. Ex vivo drug sensitivity analysis of HNSCC tumors confirmed the association between OCT4 expression and cisplatin sensitivity. Conclusion: This study introduces OCT4 immunohistochemistry as a simple and cost-effective diagnostic approach for clinical practice to identify HNSCC patients benefitting from radiosensitization by cisplatin using either full or reduced dosing.Peer reviewe

Ex vivo assessment of targeted therapies in a rare metastatic epithelial–myoepithelial carcinoma

Epithelial–myoepithelial carcinoma (EMC) is a rare subtype of salivary gland neoplasms. Since the initial description of the cancer, just over 300 cases have been reported. EMCs occupy a biphasic cellular differentiation-state defined by the constitution of two cell types representing epithelial and myoepithelial lineages, yet the functional consequence of the differentiation-state heterogeneity with respect to therapy resistance of the tumors remains unclear. The reported local recurrence rate of the cases is approximately 30%, and while distant metastases are rare, a significant fraction of these cases are reported to receive no survival benefit from radio- or chemotherapy given in addition to surgery. Moreover, no targeted therapies have been reported for these neoplasms. We report here the first use and application of ex vivo drug screening together with next generation sequencing to assess targeted treatment strategies for a rare metastatic epithelial–myoepithelial carcinoma. Results of the ex vivo drug screen demonstrate significant differential therapeutic sensitivity between the epithelial and myoepithelial intra-tumor cell lineages suggesting that differentiation-state heterogeneity within epithelial–myoepithelial carcinomas may present an outlet to partial therapeutic responses to targeted therapies including MEK and mTOR inhibitors. These results suggest that the intra-tumor lineage composition of EMC could be an important factor to be assessed when novel treatments are being evaluated for management of metastatic EMC

Proyecto de pre factibilidad para la implementación de una planta de concreto seco premezclado

El presente estudio tiene como objetivo fundamental evaluar la factibilidad de implementar una planta de concreto seco premezclado, la cual surge a partir de ofrecer una propuesta ideal e integral, ante todo práctica y sencilla, un producto alternativo a lo ya existente, está dirigido para todas aquellas personas que requieran edificar, ampliar o remodelar sus viviendas. En el capítulo inicial se efectuaron estudios en donde se determinaron los antecedentes relacionados con el proyecto así como también fundamentos teóricos importantes para el respaldo conceptual del desarrollo del proyecto. El desarrollo del estudio y análisis del mercado se determinó que existe demanda potencial con relación a la oferta existente. Además se analizó la competencia y se plantearon las estrategias de comercialización que deben realizarse para la introducción del producto. En cuanto al estudio técnico (ingeniería del proyecto), se determinó el tamaño, ubicación, infraestructura, distribución de las áreas y espacios a través de métodos como Guerchet, factores ponderados, análisis de proximidad entre aéreas, así como también procesos de generación del producto a través de los diagramas de flujo , operaciones y análisis de procesos , asimismo requerimiento de materiales , personal , entre otros. Por último la inversión que necesita el proyecto, se especifica en el capítulo estudio financiero, detallados en los estados financieros como: estado de resultados, flujo caja; éste último sirviendo de base para la evaluación financiera correspondiente; asimismo el punto de equilibrio y el análisis de sensibilidad, estudio que permite determinar si el proyecto es viable y rentable en el tiempo

Associations of reported bruxism with insomnia and insufficient sleep symptoms among media personnel with or without irregular shift work

<p>Abstract</p> <p>Background</p> <p>The aims were to investigate the prevalence of perceived sleep quality and insufficient sleep complaints, and to analyze whether self-reported bruxism was associated with perceptions of sleep, and awake consequences of disturbed sleep, while controlling confounding factors relative to poor sleep.</p> <p>Methods</p> <p>A standardized questionnaire was mailed to all employees of the Finnish Broadcasting Company with irregular shift work (n = 750) and to an equal number of randomly selected controls in the same company with regular eight-hour daytime work.</p> <p>Results</p> <p>The response rate in the irregular shift work group was 82.3% (56.6% men) and in the regular daytime work group 34.3% (46.7% men). Self-reported bruxism occurred frequently (often or continually) in 10.6% of all subjects. Altogether 16.8% reported difficulties initiating sleep (DIS), 43.6% disrupted sleep (DS), and 10.3% early morning awakenings (EMA). The corresponding figures for non-restorative sleep (NRS), tiredness, and sleep deprivation (SLD) were 36.2%, 26.1%, and 23.7%, respectively. According to logistic regression, female gender was a significant independent factor for all insomnia symptoms, and older age for DS and EMA. Frequent bruxism was significantly associated with DIS (p = 0.019) and DS (p = 0.021). Dissatisfaction with current work shift schedule and frequent bruxism were both significant independent factors for all variables describing insufficient sleep consequences.</p> <p>Conclusion</p> <p>Self-reported bruxism may indicate sleep problems and their adherent awake consequences in non-patient populations.</p

A cell spot microarray method for production of high density siRNA transfection microarrays

Peer reviewe