Response to the oncology drug gemcitabine may be variable in part due to genetic differences in the enzymes and transporters responsible for its metabolism and disposition. The aim of our in-silico ...study was to identify gene variants significantly associated with gemcitabine response that may help to personalize treatment in the clinic.
We analyzed two independent data sets: (a) genotype data from NCI-60 cell lines using the Affymetrix DMET 1.0 platform combined with gemcitabine cytotoxicity data in those cell lines, and (b) genome-wide association studies (GWAS) data from 351 pancreatic cancer patients treated on an NCI-sponsored phase III clinical trial. We also performed a subset analysis on the GWAS data set for 135 patients who were given gemcitabine+placebo. Statistical and systems biology analyses were performed on each individual data set to identify biomarkers significantly associated with gemcitabine response.
Genetic variants in the ABC transporters (ABCC1, ABCC4) and the CYP4 family members CYP4F8 and CYP4F12, CHST3, and PPARD were found to be significant in both the NCI-60 and GWAS data sets. We report significant association between drug response and variants within members of the chondroitin sulfotransferase family (CHST) whose role in gemcitabine response is yet to be delineated.
Biomarkers identified in this integrative analysis may contribute insights into gemcitabine response variability. As genotype data become more readily available, similar studies can be conducted to gain insights into drug response mechanisms and to facilitate clinical trial design and regulatory reviews.
Research in proteomics is the next step after genomics in understanding life processes at the molecular level. In the largest sense proteomics encompasses knowledge of the structure, function and ...expression of all proteins in the biochemical or biological contexts of all organisms. Since that is an impossible goal to achieve, at least in our lifetimes, it is appropriate to set more realistic, achievable goals for the field. Up to now, primarily for reasons of feasibility, scientists have tended to concentrate on accumulating information about the nature of proteins and their absolute and relative levels of expression in cells (the primary tools for this have been 2D gel electrophoresis and mass spectrometry). Although these data have been useful and will continue to be so, the information inherent in the broader definition of proteomics must also be obtained if the true promise of the growing field is to be realized. Acquiring this knowledge is the challenge for researchers in proteomics and the means to support these endeavors need to be provided. An attempt has been made to present the major issues confronting the field of proteomics and two clear messages come through in this report. The first is that the mandate of proteomics is and should be much broader than is frequently recognized. The second is that proteomics is much more complicated than sequencing genomes. This will require new technologies but it is highly likely that many of these will be developed. Looking back 10 to 20 years from now, the question is: Will we have done the job wisely or wastefully? This report summarizes the presentations made at a symposium at the National Academy of Sciences on February 25, 2002.
The aim of this study was to perform comparative analysis of multiple public datasets of gene expression in order to identify common genes as potential prognostic biomarkers. Additionally, the study ...sought to identify biological processes and pathways that are most significantly associated with early distant metastases (<5 years) in women with estrogen receptor-positive (ER+) breast tumors. Datasets from three published studies were selected for in silico analysis of gene expression profiles of ER+ breast cancer, using time to distant metastasis as the clinical endpoint. A subset of 44 differently expressed genes (DEGs) was found common to all three studies and characterized by mitotic checkpoint genes and pathways that regulate mitotic spindle and chromosome dynamics. DEG promoter regions were enriched with NFY binding sites. Analysis of miRNA target sites identified significant enrichment of miR-192, miR-193B, and miR-16–1 targets. Aberrant mitotic regulation could drive increased genomic instability leading to a progression towards an early onset metastatic phenotype. The relative importance of mitotic instability may reflect the clinical utility of mitotic poisons in metastatic breast cancer, including poisons such as the taxanes, epothilones, and vinca alkaloids.
Triple Negative Breast Cancer (TNBC), a clinically aggressive subtype of breast cancer, disproportionately affects African American (AA) women when compared to non-Hispanic Whites (NHW). ...MiRNAs(miRNAs) play a critical role in these tumors, through the regulation of cancer driver genes. In this study, our goal was to characterize and compare the patterns of miRNA expression in TNBC of AA (n = 27) and NHW women (n = 30). A total of 256 miRNAs were differentially expressed between these groups, and distinct from the ones observed in their respective non-TNBC subtypes. Fifty-five of these miRNAs were mapped in cytobands carrying copy number alterations (CNAs); 26 of them presented expression levels concordant with the observed CNAs. Receiving operating characteristic (ROC) analysis showed a good power (AUC ≥ 0.80; 95% CI) for over 65% of the individual miRNAs and a high combined power with superior sensitivity and specificity (AUC = 0.88 (0.78-0.99); 95% CI) of the 26 miRNA panel in discriminating TNBC between these populations. Subsequent miRNA target analysis revealed their involvement in the interconnected PI3K/AKT, MAPK and insulin signaling pathways. Additionally, three miRNAs of this panel were associated with early age at diagnosis. Altogether, these findings indicated that there are different patterns of miRNA expression between TNBC of AA and NHW women and that their mapping in genomic regions with high levels of CNAs is not merely physical, but biologically relevant to the TNBC phenotype. Once validated in distinct cohorts of AA women, this panel can potentially represent their intrinsic TNBC genome signature.
The aim of this study was to perform comparative analysis of multiple public datasets of gene expression in order to identify common genes as potential prognostic biomarkers. Additionally, the study ...sought to identify biological processes and pathways that are most significantly associated with early distant metastases (<5 years) in women with estrogen receptor-positive (ER+) breast tumors. Datasets from three published studies were selected for in silico analysis of gene expression profiles of ER+ breast cancer, using time to distant metastasis as the clinical endpoint. A subset of 44 differently expressed genes (DEGs) was found common to all three studies and characterized by mitotic checkpoint genes and pathways that regulate mitotic spindle and chromosome dynamics. DEG promoter regions were enriched with NFY binding sites. Analysis of miRNA target sites identified significant enrichment of miR-192, miR-193B, and miR-16–1 targets. Aberrant mitotic regulation could drive increased genomic instability leading to a progression towards an early onset metastatic phenotype. The relative importance of mitotic instability may reflect the clinical utility of mitotic poisons in metastatic breast cancer, including poisons such as the taxanes, epothilones, and vinca alkaloids.
The E1 and E2 proteins encoded by papillomaviruses are required for viral DNA replication. Although E1 is the replication initiator protein, previous studies have shown that the full-length E1 ...protein binds to the origin weakly and with low sequence specificity. The E2 protein facilitates binding of the E1 protein to the origin, triggering the initiation of replication. The E1 protein contains ATPase, helicase and DNA unwinding activities. In vivo studies with mucosal human papillomavirus (HPV) types 11 and 18 have shown that while E1 is absolutely essential for replication, the E1 binding site is dispensable. However, both the E2 protein and E2 binding sites are required for their replication. In contrast to these HPVs, transient replication of HPV type 1, which infects cutaneous tissue, requires only the viral E1 protein and E1 binding site. To understand the basis for these differences, we have overexpressed and purified the HPV-1 E1 and E2 proteins and studied their biochemical properties. The purified E1 protein was shown to have an ATPase activity with a very low K(m) value, similar to that of the SV40 large T antigen. The E1 protein bound to the HPV-1 origin in the absence of the E2 protein and without the use of any cross-linking agents. Our results suggest that the ability of the HPV-1 E1 protein to initiate DNA replication in vivo in the absence of the E2 protein may be due to its stable interaction with the HPV-1 origin.
Replication of most papillomaviruses (PVs) requires the viral-encoded E1 and E2 proteins that bind to the origin of replication (ori) containing the E1- and E2-binding sites and help recruit host ...replication factors during the initiation of DNA replication. We studied the ability of heterologous E1 and E2 proteins to interact in vivo and support replication, using the human papillomavirus (HPV) types 1a and 18 as model systems. The E1 protein of HPV-1a in combination with HPV-18 E2 supported high-level replication of various ori plasmids. In contrast, the HPV-18 E1 protein interacted weakly with HPV-1a E2 during the replication of ori plasmids. We have previously shown that the E1 protein of HPV-1a alone is sufficient for replication of HPV-1a ori plasmids, whereas HPV-18 replication requires both the E1 and E2 proteins. However, in the latter case, E2-binding sites alone in the absence of the E1-binding site can function as the minimal ori. Based on the above observations, we generated hybrids between HPV-1a and HPV-18 E1 proteins in an effort to identify their “replication specificity” domains using a transient replication assay. These hybrids were also used to localize the domains in the E1 proteins that are involved in their functional interaction with the E2 protein during replication. Our results suggest that the “replication specificity” and functional E2 interaction domains of the HPV-1a and HPV-18 E1 proteins are located in their carboxyl-terminal halves.
Due to the size and complexity of human chromosomes, viral systems have proven to be good models to elucidate the regulatory mechanisms and the proteins involved in DNA replication. Human ...Papillomaviruses (HPVs) are especially useful models since they replicate their DNA in synchrony with the host cell cycle and only two viral proteins, E1 and E2, along with the host proteins are required for replication of the viral DNA. The reliance on the host proteins for viral replication has facilitated the identification of cellular proteins involved in replication such as DNA polymerase α/primase, various cyclins/cdks and transcription factors which also function for replication enhancement. However, the interactions of these cellular and viral replication proteins with each other and with the DNA is complex and not well understood. Protein-protein and DNA-protein interactions are likely to be involved in the specificity and timing of replication. We have been investigating the specificity and requirements for the E1 and E2 proteins in the replication of HPV types 1a and 18. We have previously shown that the E1 protein of HPV-1a is sufficient for the transient replication of HPV-1a origin plasmids, although E2 significantly stimulates replication. Furthermore, a plasmid containing only the E1 binding site is capable of replication, showing that the E2 binding sites are dispensable for HPV-1a replication. In the case of HPV-18 both the E1 and E2 proteins are required for replication, however, two E2 binding sites alone can support replication. This suggests that E2 is involved in targeting E1 to the DNA and that E1 does not have a strict requirement for its binding site to initiate replication. We are interested in identifying the domains of the HPV E1 proteins that form the molecular basis for the differences in their “replication specificity” and interaction with the E2 protein. The approach used to study domain structure was to construct hybrid E1 proteins containing various portions of HPV-1a and 18 E1. Plasmids expressing the hybrid E1 proteins were used to study the transient replication of HPV-1a and HPV-18 origin plasmids in the presence of various E2 proteins in C-33A cells (HPV-negative). The hybrid E1 proteins were analyzed in the replication assays by two criteria: (1) what “replication specificity” did they possess and (2) which E2 protein did they appear to interact best with to support replication. Hybrid proteins containing the C-terminal portion from HPV-18 E1supported replication of a plasmid containing only E2 binding sites similar to that seen with HPV-18 E1. Hybrid proteins containing the C-terminal half from HPV-1a E1 replicated an origin plasmid in the absence of E2 as seen with HPV-1a E1. Additionally, 3 hybrid proteins containing the C-terminal regions from HPV-1a E1 were able to bind to an HPV-1 a origin fragment in the absence of E2 in band shift assays. These data suggest that the “replication specificity” domain of the HPV-1a E1 protein is located between amino acids 307–399, while that for the HPV-18 E1 is located between amino acids 354–442. Based on interaction of the various hybrid E1 proteins with HPV-1a or 18 E2 proteins during replication, we speculate that the E2 interaction domain of HPV-1a E1 encompasses a larger C-terminal domain including amino acids 307–612 while that of HPV-18 is present between amino acids 354–657.
Middle East respiratory syndrome coronavirus (MERS-CoV) is the causative agent of a severe respiratory disease associated with more than 2468 human infections and over 851 deaths in 27 countries ...since 2012. There are no approved treatments for MERS-CoV infection although a combination of lopinavir, ritonavir and interferon beta (LPV/RTV-IFNb) is currently being evaluated in humans in the Kingdom of Saudi Arabia. Here, we show that remdesivir (RDV) and IFNb have superior antiviral activity to LPV and RTV in vitro. In mice, both prophylactic and therapeutic RDV improve pulmonary function and reduce lung viral loads and severe lung pathology. In contrast, prophylactic LPV/RTV-IFNb slightly reduces viral loads without impacting other disease parameters. Therapeutic LPV/RTV-IFNb improves pulmonary function but does not reduce virus replication or severe lung pathology. Thus, we provide in vivo evidence of the potential for RDV to treat MERS-CoV infections.
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