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A novel assay was developed for Daudi cells in which the antiviral (AV) and antiproliferative (AP) activities of interferon (IFN) can be measured simultaneously. Using this novel assay, conditions allowing IFN AV protection but no growth inhibition were identified and selected. Daudi cells were treated under these conditions, and gene expression microarray analyses were performed. The results of the analysis identified 25 genes associated with IFN-α AV activity. Upregulation of 23 IFN-induced genes was confirmed by using reverse transcription-PCR. Of 25 gene products, 17 were detected by Western blotting at 24 h. Of the 25 genes, 10 have not been previously linked to AV activity of IFN-α. The most upregulated gene was IFIT3 (for IFN-induced protein with tetratricopeptide repeats 3). The results from antibody neutralizing experiments suggested an association of the identified genes with IFN-α AV activity. This association was strengthened by results from IFIT3-small interfering RNA transfection experiments showing decreased expression of IFIT3 and a reduction in the AV activity induced by IFN-α. Overexpression of IFIT3 resulted in a decrease of virus titer. Transcription of AV genes after the treatment of cells with higher concentrations of IFN having an AP effect on Daudi cells suggested pleiotropic functions of identified gene products.

Human ES (hES) cell lines have only recently been generated, and differences between human and mouse ES cells have been identified. In this manuscript we describe the properties of two human ES cell lines, BG01 and BG02. By immunocytochemistry and reverse transcription polymerase chain reaction, undifferentiated cells expressed markers that are characteristic of ES cells, including SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and OCT-3/4. Both cell lines were readily maintained in an undifferentiated state and could differentiate into cells of all three germ layers, as determined by expression of beta-tubulin III neuron-specific molecule (ectoderm), cardiac troponin I (cardiomyocytes, mesoderm), and alpha-fetoprotein (endoderm). A large-scale microarray (16,659 genes) analysis identified 373 genes that were expressed at three-fold or higher levels in undifferentiated BG01 and BG02 cells as compared with pooled human RNA. Ninety-two of these genes were also highly expressed in four other hES lines (TE05, GE01, GE09, and pooled samples derived from GE01, GE09, and GE07). Included in the list are genes involved in cell signaling and development, metabolism, transcription regulation, and many hypothetical proteins. Two focused arrays designed to examine transcripts associated with stem cells and with the transforming growth factor-beta superfamily were employed to examine differentially expressed genes. Several growth factors, receptors, and components of signaling pathways that regulate embryonic development, in particular the nodal signaling pathway, were detected in both BG01 and BG02. These data provide a detailed characterization and an initial gene expression profile for the BG01 and BG02 human ES cell lines.

Clinical applications of human interferon (IFN)-α have met with varying degrees of success. Nevertheless, key molecules in cell viability regulated by IFN-α have not been clearly identified. Our previous study indicated that IFN (α, β and ω) receptor (IFNAR) 1/2- and IFN regulatory factor (IRF) 9-RNA interference (RNAi) completely restored cell viability and transcription of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) following IFN-α treatment, in human ovarian adenocarcinoma OVCAR3 cells sensitive to IFN-α. In the present study, IFNAR1/2- and IRF9-RNAi inhibited the gene expression of TRAIL, but not of Fas ligand (FasL), following IFN-α treatment. In fact, TRAIL but not FasL inhibited the viability of OVCAR3 cells. IFN-α notably up-regulated the levels of TRAIL protein in the supernatant and on the membrane of OVCAR3 cells. Following TRAIL signaling, Caspase 8 inhibitor and BH3 interacting domain death agonist (BID)-RNAi significantly restored cell viability in response to IFN-α and TRAIL in OVCAR3 cells. Furthermore, BID-RNAi prevented both IFN-α and TRAIL from collapsing the mitochondrial membrane potential (ΔΨm). Finally, we provided important evidence that BID overexpression led to significant inhibition of cell viability following IFN-α or TRAIL treatments in human lung carcinoma A549 cells resistant to IFN-α. Thus, this study suggests that BID is crucial for cell viability regulated by IFN-α which can induce mitochondria-mediated apoptosis, indicating a notable potential to be a targeted therapy for IFN-α resistant tumors.

A number of tumors are still resistant to the antiproliferative activity of human interferon (IFN)-alpha. The Janus kinases/Signal Transducers and Activators of Transcription (JAK-STAT) pathway plays an important role in initial IFN signaling. To enhance the antiproliferative activity of IFN-alpha, it is important to elucidate which factors in the JAK-STAT pathway play a key role in eliciting this activity. In human ovarian adenocarcinoma OVCAR3 cells sensitive to both IFN-alpha and IFN-gamma, only IFN regulatory factor 9 (IRF9)-RNA interference (RNAi) completely inhibited the antiproliferative activity of IFN-alpha among the intracellular JAK-STAT pathway factors. Conversely, Stat1-RNAi did not inhibit the antiproliferative activity of IFN-alpha, whereas it partially inhibited that of IFN-gamma. As a cell death pathway, it is reported that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis through TRAIL-receptor (R) 1 and TRAIL-R2. In IFN-alpha-treated OVCAR3 cells, IRF9-RNAi inhibited transcription of TRAIL whereas Stat1-RNAi did not, suggesting that the transcription of TRAIL induced by IFN-alpha predominantly required IRF9. Furthermore, IFN-stimulated response element-like motifs of TRAIL bound to IFN-stimulated gene factor 3 (ISGF3) complex after IFN-alpha treatment. Subsequently, TRAIL-R2-RNAi inhibited both antiproliferative activities of IFN-alpha and TRAIL, suggesting that TRAIL-R2 mediated both IFN-alpha and TRAIL signals to elicit their antiproliferative activities. Finally, IRF9 overexpression facilitated IFN-alpha-induced apoptosis in T98G (human glioblastoma multiforme) cells, which were resistant to IFN-alpha. Thus, this study suggests that IRF9 is the key factor for eliciting the antiproliferative activity of IFN-alpha and TRAIL may be one of the potential mediators.

Background The identification of molecular pathways of differentiation of embryonic stem cells (hESC) is critical for the development of stem cell based medical therapies. In order to identify biomarkers and potential regulators of the process of differentiation, a high quality microarray containing 16,659 seventy base pair oligonucleotides was used to compare gene expression profiles of undifferentiated hESC lines and differentiating embryoid bodies. Results Previously identified "stemness" genes in undifferentiated hESC lines showed down modulation in differentiated cells while expression of several genes was induced as cells differentiated. In addition, a subset of 194 genes showed overexpression of greater than ≥ 3 folds in human embryoid bodies (hEB). These included 37 novel and 157 known genes. Gene expression was validated by a variety of techniques including another large scale array, reverse transcription polymerase chain reaction, focused cDNA microarrays, massively parallel signature sequencing (MPSS) analysis and immunocytochemisty. Several novel hEB specific expressed sequence tags (ESTs) were mapped to the human genome database and their expression profile characterized. A hierarchical clustering analysis clearly depicted a distinct difference in gene expression profile among undifferentiated and differentiated hESC and confirmed that microarray analysis could readily distinguish them. Conclusion These results present a detailed characterization of a unique set of genes, which can be used to assess the hESC differentiation.

Human embryonic stem (huES) cells have the ability to differentiate into a variety of cell lineages and potentially provide a source of differentiated cells for many therapeutic uses. However, little is known about the mechanism of differentiation of huES cells and factors regulating cell development. We have used high-quality microarrays containing 16 659 seventy–base pair oligonucleotides to examine gene expression in 6 of the 11 available huES cell lines. Expression was compared against pooled RNA from multiple tissues (universal RNA) and genes enriched in huES cells were identified. All 6 cell lines expressed multiple markers of the undifferentiated state and shared significant homology in gene expression (overall similarity coefficient > 0.85).A common subset of 92 genes was identified that included Nanog, GTCM-1, connexin 43 (GJA1), oct-4, and TDGF1 (cripto). Gene expression was confirmed by a variety of techniques including comparison with databases, reverse transcriptase–polymerase chain reaction, focused cDNA microarrays, and immunocytochemistry. Comparison with published “stemness” genes revealed a limited overlap, suggesting little similarity with other stem cell populations. Several novel ES cell–specific expressed sequence tags were identified and mapped to the human genome. These results represent the first detailed characterization of undifferentiated huES cells and provide a unique set of markers to profile and better understand the biology of huES cells. (Blood. 2004;103: 2956-2964)

As the quality of microarrays is critical to successful experiments for data consistency and validity, a reliable and convenient quality control method is needed. We describe a systematic quality control method for large-scale genome oligonucleotide arrays. This method is comprised of three steps to assess the quality of printed arrays. The first step involves assessment of the autofluorescence property of DNA. This step is convenient, quick to perform, and allowed reuse of every array. The second step involves hybridization of arrays with Cy3-labeled 9-mer oligonucleotide target to assess the quality and stability of oligonucleotides. Because this step consumed arrays, one or two arrays from each batch were used to complement the quality control data from autofluorescence. The third step involves hybridization of arrays from every batch with transcripts derived from two cell lines to assess data consistency. These hybridizations were able to distinguish two closely related tissue samples by identifying a cluster of 20 genes that were differently expressed in U87MG and T98G glioblastoma cell lines. In addition, we standardized two parameters that significantly enhanced the quality of arrays. We found that longer pin contact time and crosslinking oligonucleotides at 400 mJ/cm(2) were optimal for the highest hybridization intensity. Taken together, these results indicate that the quality of spotted oligonucleotide arrays should be assessed by at least two methods, autofluorescence and 9-mer hybridization before arrays are used for hybridization experiments.