Cellular Model of Malignant Transformation of Primary Human Astrocytes Induced by Deadhesion/Readhesion Cycles.

Astrocytoma is the most common and aggressive tumor of the central nervous system. Genetic and environmental factors, bacterial infection, and several other factors are known to be involved in gliomagenesis, although the complete underlying molecular mechanism is not fully understood. Tumorigenesis is a multistep process involving initiation, promotion, and progression. We present a human model of malignant astrocyte transformation established by subjecting primary astrocytes from healthy adults to four sequential cycles of forced anchorage impediment (deadhesion). After limiting dilution of the surviving cells obtained after the fourth deadhesion/readhesion cycle, three clones were randomly selected, and exhibited malignant characteristics, including increased proliferation rate and capacity for colony formation, migration, and anchorage-independent growth in soft agar. Functional assay results for these clonal cells, including response to temozolomide, were comparable to U87MG-a human glioblastoma-derived cell lineage-reinforcing malignant cell transformation. RNA-Seq analysis by next-generation sequencing of the transformed clones relative to the primary astrocytes revealed upregulation of genes involved in the PI3K/AKT and Wnt/ß-catenin signaling pathways, in addition to upregulation of genes related to epithelial-mesenchymal transition, and downregulation of genes related to aerobic respiration. These findings, at a molecular level, corroborate the change in cell behavior towards mesenchymal-like cell dedifferentiation. This linear progressive model of malignant human astrocyte transformation is unique in that neither genetic manipulation nor treatment with carcinogens are used, representing a promising tool for testing combined therapeutic strategies for glioblastoma patients, and furthering knowledge of astrocytoma transformation and progression.

Kinome siRNA screen identifies novel cell-type specific dengue host target genes.

Dengue is a global emerging infectious disease, with no specific treatment available. To identify novel human host cell targets important for dengue virus infection and replication, an image-based high-throughput siRNA assay screening of a human kinome siRNA library was conducted using human hepatocyte cell line Huh7 infected with a recent dengue serotype 2 virus isolate BR DEN2 01-01. In the primary siRNA screening of 779 kinase-related genes, knockdown of 22 genes showed a reduction in DENV-2 infection. Conversely, knockdown of 8 genes enhanced viral infection. To assess host cell specificity, the confirmed hits were tested in the DENV-infected monocytic cell line U937. While the expression of EIF2AK3, ETNK2 and SMAD7 was regulated in both cell lines after infection, most kinases were hepatocyte-specific. Monocytic cells represent initial targets of infection and an antiviral treatment targeting these cells is probably most effective to reduce initial viral load. In turn, infection of the liver could contribute to pathogenesis, and the novel hepatocyte-specific human targets identified here could be important for dengue infection and pathogenesis.

Identification of novel compounds inhibiting chikungunya virus-induced cell death by high throughput screening of a kinase inhibitor library.

Chikungunya virus (CHIKV) is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing ≥50% inhibition property against CHIKV at 10 µM were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415), one pyrrolopyridine (CND0545) and one thiazol-carboxamide (CND3514) inhibited CHIKV-associated cell death in a dose-dependent manner, with EC50 values between 2.2 µM and 7.1 µM. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against CHIKV having a novel antiviral activity--inhibition of virus-induced CPE--likely by targeting kinases involved in apoptosis.