BackgroundGene expression profiling is being widely applied in cancer research to identify biomarkers for clinical endpoint prediction. Since RNA-seq provides a powerful tool for transcriptome-based applications beyond the limitations of microarrays, we sought to systematically evaluate the performance of RNA-seq-based and microarray-based classifiers in this MAQC-III/SEQC study for clinical endpoint prediction using neuroblastoma as a model.ResultsWe generate gene expression profiles from 498 primary neuroblastomas using both RNA-seq and 44 k microarrays. Characterization of the neuroblastoma transcriptome by RNA-seq reveals that more than 48,000 genes and 200,000 transcripts are being expressed in this malignancy. We also find that RNA-seq provides much more detailed information on specific transcript expression patterns in clinico-genetic neuroblastoma subgroups than microarrays. To systematically compare the power of RNA-seq and microarray-based models in predicting clinical endpoints, we divide the cohort randomly into training and validation sets and develop 360 predictive models on six clinical endpoints of varying predictability. Evaluation of factors potentially affecting model performances reveals that prediction accuracies are most strongly influenced by the nature of the clinical endpoint, whereas technological platforms (RNA-seq vs. microarrays), RNA-seq data analysis pipelines, and feature levels (gene vs. transcript vs. exon-junction level) do not significantly affect performances of the models.ConclusionsWe demonstrate that RNA-seq outperforms microarrays in determining the transcriptomic characteristics of cancer, while RNA-seq and microarray-based models perform similarly in clinical endpoint prediction. Our findings may be valuable to guide future studies on the development of gene expression-based predictive models and their implementation in clinical practice.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0694-1) contains supplementary material, which is available to authorized users.

BACKGROUND: Glycosylated proteins play important roles in cell-to-cell interactions, immunosurveillance, and a variety of receptor-mediated and specific protein functions through a highly complex repertoire of glycan structures. Aberrant glycosylation has been implicated in cancer for many years.

Topoisomerase I cleavage complexes can be induced by a variety of DNA damages and by the anticancer drug camptothecin. We have developed a ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by topoisomerase I cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level. We found that conversion of topoisomerase I cleavage complexes into replicationmediated DNA double-strand breaks was only detectable on the leading strand for DNA synthesis, which suggests an asymmetry in the way that topoisomerase I cleavage complexes are metabolized on the two arms of a replication fork. Extension by Taq DNA polymerase was not required for ligation to the LM-PCR primer, indicating that the 3 DNA ends are extended by DNA polymerase in vivo closely to the 5 ends of the topoisomerase I cleavage complexes. These findings suggest that the replication-mediated DNA double-strand breaks generated at topoisomerase I cleavage sites are produced by replication runoff. We also found that the 5 ends of these DNA double-strand breaks are phosphorylated in vivo, which suggests that a DNA 5 kinase activity acts on the double-strand ends generated by replication runoff. The replication-mediated DNA doublestrand breaks were rapidly reversible after cessation of the topoisomerase I cleavage complexes, suggesting the existence of efficient repair pathways for removal of topoisomerase I-DNA covalent adducts in ribosomal DNA.DNA topoisomerases are ubiquitous enzymes that regulate the topological state of DNA. They participate in essential cellular processes, including replication, transcription, chromosome segregation, and recombination (22,34,71). Eukaryotic DNA topoisomerase I (top1) acts as a monomer, and its catalytic activity can be divided into four steps (61): (i) binding of the enzyme to duplex DNA, (ii) single-stranded DNA cleavage by a transesterification reaction in which a top1 tyrosinehydroxyl group becomes covalently linked to the 3Ј phosphate of a DNA phosphodiester bond to generate a 5Ј-hydroxyl DNA terminus, (iii) DNA relaxation by controlled rotation around the intact DNA strand (61); and (iv) religation of the cleaved DNA by nucleophilic attack from the 5Ј-hydroxyl DNA end and dissociation of the top1 tyrosyl residue from the 3Ј end. The topoisomerase-linked DNA breaks are commonly referred to as cleavage complexes (22,34,71). Under physiological conditions, they are short-lived catalytic intermediates.A number of physiological and environmental DNA modifications can inhibit top1 by inducing top1 cleavage complexes. These include DNA mismatches or abasic sites (37, 48, 73), oxidative base damage (47), base alkylation and carcinogenic adducts (44, 66), UV photoproducts (50, 62), and DNA breaks (11, 45). Trapping of top1 cleavage complexes is also the primary mechanism of action of camptothecin (CPT), a potent anticancer agent which reversibly inhibits the religation step of the top1 catalytic cycle (25,29,39,40). The cytotoxicity of top1 cleavage complexes is atte...

A sedimentable complex of enzymes for DNA synthesis was partially purified from the combined low-salt nuclear extract-postmicrosomal supernatant solution of HeLa cell homogenates by poly(ethylene glycol) precipitation in the presence of 2 M KCl, discontinuous gradient centrifugation, Q-Sepharose chromatography, and velocity gradient centrifugation. In addition to the previously described 640-kDa multiprotein DNA polymerase alpha-primase complex [Vishwanatha et al. (1986) J. Biol. Chem. 261, 6619-6628], the enzyme complex also has associated topoisomerase I, DNA-dependent ATPase, RNase H, DNA ligase, a simian virus 40 origin recognition, dA/dT sequence binding protein [Malkas & Baril (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 70-74], and proliferating cell nuclear antigen. Essentially all of the T antigen dependent simian virus 40 in vitro replication activity in the combined nuclear extract-postmicrosomal supernatant solution resides with the sedimentable complex of enzymes for DNA synthesis. Sedimentation analysis on a 10-35% glycerol gradient in the presence of 0.5 M KCl indicates that the enzyme complex is 21S. The associated enzymes for DNA synthesis and in vitro simian virus 40 replication activity cofractionate throughout the purification of the 21S complex. The DNA polymerase and in vitro simian virus 40 replication activities are both inhibited by monoclonal antibody (SJK 132-20) to human DNA polymerase alpha and by 5-10 microM butylphenyl-dGTP, indicating that the association of DNA polymerase alpha with the 21S enzyme complex is essential for the initiation of SV40 DNA replication in vitro.

Proliferating cell nuclear antigen (PCNA), a potential anticancer target, forms a homotrimer and is required for DNA replication and numerous other cellular processes. The purpose of this study was to identify novel small molecules that modulate PCNA activity to affect tumor cell proliferation. An in silico screen of a compound library against a crystal structure of PCNA and a subsequent structural similarity search of the ZINC chemical database were carried out to derive relevant docking partners. Nine compounds, termed PCNA inhibitors (PCNA-Is), were selected for further characterization. PCNA-I1 selectively bound to PCNA trimers with a dissociation constant (K d ) of ϳ0.2 to 0.4 M. PCNA-Is promoted the formation of SDSrefractory PCNA trimers. PCNA-I1 dose-and time-dependently reduced the chromatin-associated PCNA in cells. Consistent with its effects on PCNA trimer stabilization, PCNA-I1 inhibited the growth of tumor cells of various tissue types with an IC 50 of ϳ0.2 M, whereas it affected the growth of nontransformed cells at significantly higher concentrations (IC 50 , ϳ1.6 M). Moreover, uptake of BrdU was dose-dependently reduced in cells treated with PCNA-I1. Mechanistically the PCNA-Is mimicked the effect of PCNA knockdown by siRNA, inducing cancer cell arrest at both the S and G 2 /M phases. Thus, we have identified a class of compounds that can directly bind to PCNA, stabilize PCNA trimers, reduce PCNA association with chromatin, and inhibit tumor cell growth by inducing a cell cycle arrest. They are valuable tools in studying PCNA function and may be useful for future PCNA-targeted cancer therapy.