Replication and Virus-Induced Transcriptome of HAdV-5 in Normal Host Cells versus Cancer Cells - Differences of Relevance for Adenoviral Oncolysis

Adenoviruses (Ads), especially HAdV-5, have been genetically equipped with tumor-restricted replication potential to enable applications in oncolytic cancer therapy. Such oncolytic adenoviruses have been well tolerated in cancer patients, but their anti-tumor efficacy needs to be enhanced. In this regard, it should be considered that cancer cells, dependent on their tissue of origin, can differ substantially from the normal host cells to which Ads are adapted by complex virus-host interactions. Consequently, viral replication efficiency, a key determinant of oncolytic activity, might be suboptimal in cancer cells. Therefore, we have analyzed both the replication kinetics of HAdV-5 and the virus-induced transcriptome in human bronchial epithelial cells (HBEC) in comparison to cancer cells. This is the first report on genome-wide expression profiling of Ads in their native host cells. We found that E1A expression and onset of viral genome replication are most rapid in HBEC and considerably delayed in melanoma cells. In squamous cell lung carcinoma cells, we observed intermediate HAdV-5 replication kinetics. Infectious particle production, viral spread and lytic activity of HAdV-5 were attenuated in melanoma cells versus HBEC. Expression profiling at the onset of viral genome replication revealed that HAdV-5 induced the strongest changes in the cellular transcriptome in HBEC, followed by lung cancer and melanoma cells. We identified prominent regulation of genes involved in cell cycle and DNA metabolism, replication and packaging in HBEC, which is in accord with the necessity to induce S phase for viral replication. Strikingly, in melanoma cells HAdV-5 triggered opposing regulation of said genes and, in contrast to lung cancer cells, no weak S phase induction was detected when using the E2F promoter as reporter. Our results provide a rationale for improving oncolytic adenoviruses either by adaptation of viral infection to target tumor cells or by modulating tumor cell functions to better support viral replication

The RNA-binding protein TbDRBD3 regulates the stability of a specific subset of mRNAs in trypanosomes

In trypanosomes, the apparent lack of regulation of RNA polymerase II-dependent transcription initiation poses a challenge to understand how these eukaryotes adjust gene expression to adapt to the contrasting environments they find during their life cycles. Evidence so far indicates that mRNA turnover and translation are the major control points in which regulation is exerted in trypanosomes. However, very little is known about which proteins are involved, and how do they regulate the abundance and translation of different mRNAs in different life stages. In this work, an RNA-binding protein, TbDRBD3, has been identified by affinity chromatography, and its function addressed using RNA interference, microarray analysis and immunoprecipitation of mRNA–protein complexes. The results obtained indicate that TbDRBD3 binds to a subset of developmentally regulated mRNAs encoding membrane proteins, and that this association promotes the stabilization of the target transcripts. These observations raise the possibility that TbDRBD3-mRNA complexes act as a post-transcriptional operon, and provide a framework to interpret how trypanosomes regulate gene expression in the absence of transcriptional control

Microarray data warehouse allowing for inclusion of experiment annotations in statistical analysis

Motivation: Microarray technology provides access to expression levels of thousands of genes at once, producing large amounts of data. These datasets are valuable only if they are annotated by sufficiently detailed experiment descriptions. However, in many databases a substantial number of these annotations is in free-text format and not readily accessible to computer-aided analysis. Results: The Multi-Conditional Hybridization Intensity Processing System (M-CHIPS), a data warehousing concept, focuses on providing both structure and algorithms suitable for statistical analysis of a microarray database’s entire contents including the experiment annotations. It addresses the rapid growth of the amount of hybridization data, more detailed experimental descriptions, and new kinds of experiments in the future. We have developed a storage concept , a particular instance of which is an organism-specific database. Although these databases may contain different ontologies of experiment annotations, they share the same structure and therefore can be accessed by the very same statistical algorithms. Experiment ontologies have not yet reached their final shape, and standards are reduced to minimal conventions that do not yet warrant extensive description. An ontology-independent structure enables updates of annotation hierarchies during normal database operation without altering the structure

Microarray data warehouse allowing for inclusion of experiment annotations in statistical analysis

Motivation: Microarray technology provides access to expression levels of thousands of genes at once, producing large amounts of data. These datasets are valuable only if they are annotated by sufficiently detailed experiment descriptions. However, in many databases a substantial number of these annotations is in free-text format and not readily accessible to computer-aided analysis. Results: The Multi-Conditional Hybridization Intensity Processing System (M-CHIPS), a data warehousing concept, focuses on providing both structure and algorithms suitable for statistical analysis of a microarray database’s entire contents including the experiment annotations. It addresses the rapid growth of the amount of hybridization data, more detailed experimental descriptions, and new kinds of experiments in the future. We have developed a storage concept , a particular instance of which is an organism-specific database. Although these databases may contain different ontologies of experiment annotations, they share the same structure and therefore can be accessed by the very same statistical algorithms. Experiment ontologies have not yet reached their final shape, and standards are reduced to minimal conventions that do not yet warrant extensive description. An ontology-independent structure enables updates of annotation hierarchies during normal database operation without altering the structure

Functional studies on the galectin-4 promoter and its use for establishing a transcription factors array assay

In the present study, I profiled the expression pattern of different stages of six colorectal cancer and adenoma cell lines (SW1116, SW480, SW620, Co115, KM20L2, and LT97 in comparison to normal colon cell line CCD-18co). In our expression profiling data, we have found that galectin-4 was among the genes that are significantly upregulated in LT97 and KM20L2. We investigated galectin-4 upregulation as a signature of bad prognosis in colorectal cancer cell lines. In addition, we identified one possible mechanism of galectin-4 upregulation which is associated with a twin SNPs in the upstream sequence and the first intron. From the sequencing results of 115 patients, 26 out of the 115 (22.6%) were found to have the two SNPs together (ss217320370 and rs73933062), while the other patients did not carry any of these two SNPs. Therefore, we firstly showed that ss217320370 C>A at position 11572034 (a novel SNP was identified in the present study) and G>A at position 11571652 (rs73933062) are always together in the same individual (twin SNPs). Since the twin SNPs could potentially be associated with galectin-4 upregulation via deletion and insertion of new transcription factor binding sites, the twin SNPs may have medical impact in colorectal cancer patient. In parallel, another project was carried out to establish transcription factor protein. In conclusion, upregulation of galectin-4 was found to be associated with bad prognosis of colorectal cancer. In addition, two SNPs (ss217320370 and rs73933062) were found to be associated with galectin-4 upregulation, which might be referred to the changing in the binding sequence of the regulatory elements. Using galectin-4 promoter with the twin SNPs was useful in setting up TFs-array to detect DNA-protein interactions, since the microarray result was concordant with pull down-mass spectrometry analysis of galectin-4 promoter