| 1. |
- Danielsson, Frida, et al.
(författare)
-
Majority of differentially expressed genes are down-regulated during malignant transformation in a four-stage model
- 2013
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 110:17, s. 6853-6858
-
Tidskriftsartikel (refereegranskat)abstract
- The transformation of normal cells to malignant, metastatic tumor cells is a multistep process caused by the sequential acquirement of genetic changes. To identify these changes, we compared the transcriptomes and levels and distribution of proteins in a four-stage cell model of isogenically matched normal, immortalized, transformed, and metastatic human cells, using deep transcriptome sequencing and immunofluorescence microscopy. The data show that similar to 6% (n = 1,357) of the human protein-coding genes are differentially expressed across the stages in the model. Interestingly, the majority of these genes are down-regulated, linking malignant transformation to dedifferentiation. The up-regulated genes are mainly components that control cellular proliferation, whereas the down-regulated genes consist of proteins exposed on or secreted from the cell surface. As many of the identified gene products control basic cellular functions that are defective in cancers, the data provide candidates for follow-up studies to investigate their functional roles in tumor formation. When we further compared the expression levels of four of the identified proteins in clinical cancer cohorts, similar differences were observed between benign and cancer cells, as in the cell model. This shows that this comprehensive demonstration of the molecular changes underlying malignant transformation is a relevant model to study the process of tumor formation.
|
|
| 2. |
- Fagerberg, Linn, et al.
(författare)
-
Contribution of antibody-based protein profiling to the human chromosome-centric proteome project (C-HPP)
- 2013
-
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 12:6, s. 2439-2448
-
Tidskriftsartikel (refereegranskat)abstract
- A gene-centric Human Proteome Project has been proposed to characterize the human protein-coding genes in a chromosome-centered manner to understand human biology and disease. Here, we report on the protein evidence for all genes predicted from the genome sequence based on manual annotation from literature (UniProt), antibody-based profiling in cells, tissues and organs and analysis of the transcript profiles using next generation sequencing in human cell lines of different origins. We estimate that there is good evidence for protein existence for 69% (n = 13985) of the human protein-coding genes, while 23% have only evidence on the RNA level and 7% still lack experimental evidence. Analysis of the expression patterns shows few tissue-specific proteins and approximately half of the genes expressed in all the analyzed cells. The status for each gene with regards to protein evidence is visualized in a chromosome-centric manner as part of a new version of the Human Protein Atlas (www.proteinatlas.org).
|
|
| 3. |
- Georgoudaki, Anna-Maria, et al.
(författare)
-
Reprogramming Tumor-Associated Macrophages by Antibody Targeting Inhibits Cancer Progression and Metastasis
- 2016
-
Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 15:9, s. 2000-2011
-
Tidskriftsartikel (refereegranskat)abstract
- Tumors are composed of multiple cell types besides the tumor cells themselves, including innate immune cells such as macrophages. Tumor-associated macrophages (TAMs) are a heterogeneous population of myeloid cells present in the tumor microenvironment (TME). Here, they contribute to immunosuppression, enabling the establishment and persistence of solid tumors as well as metastatic dissemination. We have found that the pattern recognition scavenger receptor MARCO defines a subtype of suppressive TAMs and is linked to clinical outcome. An anti-MARCO monoclonal antibody was developed, which induces anti-tumor activity in breast and colon carcinoma, as well as in melanoma models through reprogramming-TAM-populations to a pro-inflammatory phenotype and increasing tumor immunogenicity. This anti-tumor activity is dependent on the inhibitory Fc-receptor, Fc gamma RIIB, and also enhances the efficacy of checkpoint therapy. These results demonstrate that immunotherapies using antibodies designed to modify myeloid cells of the TME represent a promising mode of cancer treatment.
|
|
| 4. |
- Klevebring, Daniel, 1981-, et al.
(författare)
-
Analysis of transcript and protein overlap in a human osteosarcoma cell line
- 2010
-
Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 11:1, s. 684-
-
Tidskriftsartikel (refereegranskat)abstract
- Background: An interesting field of research in genomics and proteomics is to compare the overlap between the transcriptome and the proteome. Recently, the tools to analyse gene and protein expression on a whole-genome scale have been improved, including the availability of the new generation sequencing instruments and high-throughput antibody-based methods to analyze the presence and localization of proteins. In this study, we used massive transcriptome sequencing (RNA-seq) to investigate the transcriptome of a human osteosarcoma cell line and compared the expression levels with in situ protein data obtained in-situ from antibody-based immunohistochemistry (IHC) and immunofluorescence microscopy (IF). Results: A large-scale analysis based on 2749 genes was performed, corresponding to approximately 13% of the protein coding genes in the human genome. We found the presence of both RNA and proteins to a large fraction of the analyzed genes with 60% of the analyzed human genes detected by all three methods. Only 34 genes (1.2%) were not detected on the transcriptional or protein level with any method. Our data suggest that the majority of the human genes are expressed at detectable transcript or protein levels in this cell line. Since the reliability of antibodies depends on possible cross-reactivity, we compared the RNA and protein data using antibodies with different reliability scores based on various criteria, including Western blot analysis. Gene products detected in all three platforms generally have good antibody validation scores, while those detected only by antibodies, but not by RNA sequencing, generally consist of more low-scoring antibodies. Conclusion: This suggests that some antibodies are staining the cells in an unspecific manner, and that assessment of transcript presence by RNA-seq can provide guidance for validation of the corresponding antibodies.
|
|
| 5. |
- Klevebring, Daniel, 1981-, et al.
(författare)
-
Automation of cDNA Synthesis and Labelling Improves Reproducibility
- 2009
-
Ingår i: Journal of Biomedicine and Biotechnology. - : Hindawi Limited. - 1110-7243 .- 1110-7251. ; 2009, s. 396808-
-
Tidskriftsartikel (refereegranskat)abstract
- Background. Several technologies, such as in-depth sequencing and microarrays, enable large-scale interrogation of genomes and transcriptomes. In this study, we asses reproducibility and throughput by moving all laboratory procedures to a robotic workstation, capable of handling superparamagnetic beads. Here, we describe a fully automated procedure for cDNA synthesis and labelling for microarrays, where the purification steps prior to and after labelling are based on precipitation of DNA on carboxylic acid-coated paramagnetic beads. Results. The fully automated procedure allows for samples arrayed on a microtiter plate to be processed in parallel without manual intervention and ensuring high reproducibility. We compare our results to a manual sample preparation procedure and, in addition, use a comprehensive reference dataset to show that the protocol described performs better than similar manual procedures. Conclusions. We demonstrate, in an automated gene expression microarray experiment, a reduced variance between replicates, resulting in an increase in the statistical power to detect differentially expressed genes, thus allowing smaller differences between samples to be identified. This protocol can with minor modifications be used to create cDNA libraries for other applications such as in-depth analysis using next-generation sequencing technologies.
|
|
| 6. |
- Klevebring, Daniel, et al.
(författare)
-
Exome sequencing of contralateral breast cancer identifies metastatic disease
- 2015
-
Ingår i: Breast Cancer Research and Treatment. - Stockholm : Karolinska Institutet, Dept of Medical Epidemiology and Biostatistics. - 0167-6806 .- 1573-7217.
-
Tidskriftsartikel (refereegranskat)abstract
- Women with contralateral breast cancer (CBC) have significantly worse prognosis compared to women with unilateral cancer. A possible explanation of the poor prognosis of patients with CBC is that in a subset of patients, the second cancer is not a new primary tumor but a metastasis of the first cancer that has potentially obtained aggressive characteristics through selection of treatment. Exome and whole-genome sequencing of solid tumors has previously been used to investigate the clonal relationship between primary tumors and metastases in several diseases. In order to assess the relationship between the first and the second cancer, we performed exome sequencing to identify somatic mutations in both first and second cancers, and compared paired normal tissue of 25 patients with metachronous CBC. For three patients, we identified shared somatic mutations indicating a common clonal origin thereby demonstrating that the second tumor is a metastasis of the first cancer, rather than a new primary cancer. Accordingly, these patients all developed distant metastasis within 3 years of the second diagnosis, compared with 7 out of 22 patients with non-shared somatic profiles. Genomic profiling of both tumors help the clinicians distinguish between true CBCs and subsequent metastases.
|
|
| 7. |
- Klevebring, Daniel, 1981-, et al.
(författare)
-
Genome-wide profiling of Populus small RNAs
- 2009
-
Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 10, s. Article number 620-
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Short RNAs, and in particular microRNAs, are important regulators of gene expression both within defined regulatory pathways and at the epigenetic scale. We investigated the short RNA (sRNA) population (18-24 nt) of the transcriptome of green leaves from the sequenced Populus trichocarpa using a concatenation strategy in combination with 454 sequencing. Results: The most abundant size class of sRNAs were 24 nt and these were generally associated with a number of classes of retrotransposons and repetitive elements. Some repetitive elements were also associated with 22 nt RNAs. We identified an sRNA hot-spot on chromosome 19, overlapping a region containing both the sex-determining loci and a major cluster of NBS-LRR genes. A number of phased siRNA loci were identified, a subset of which are predicted to target PPR and NBS-LRR disease resistance genes, classes of genes that have been significantly expanded in Populus. Additional loci enriched for sRNA production were identified. We identified 15 novel predicted microRNAs (miRNAs), including miRNA∗ sequences, and identified a novel locus that may encode a dual miRNA or a miRNA and short interfering RNAs (siRNAs). Conclusions: The short RNA population of P. trichocarpa is at least as complex as that of Arabidopsis. We provide a first genome-wide view of short RNA production for P. trichocarpa and identify new, non-conserved miRNAs.
|
|
| 8. |
- Klevebring, Daniel, 1981-, et al.
(författare)
-
In-depth transcriptome analysis reveals novel TARs and prevalent antisense transcription in human cell lines.
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Several recent studies have indicated that transcription is pervasive in regions outside of protein coding genes and that short antisense transcripts can originate from the promoter and terminator regions of genes. Here we investigate transcription of fragments longer than 200 nucleotides, focusing on antisense transcription for known protein coding genes and intergenic transcription. We find that roughly 12% to 16% of all reads that originate from promoter and terminator regions, respectively, map antisense to the gene in question. Furthermore, we detect a high number of novel transcriptionally active regions (TARs) that are generally expressed at a lower level than protein coding genes. We also investigate the correlation between RNA-seq data and microarray data and conclude that the correlation is dependant on gene length such that longer genes show a better correlation.
|
|
| 9. |
- Klevebring, Daniel, et al.
(författare)
-
In-Depth Transcriptome Analysis Reveals Novel TARs and Prevalent Antisense Transcription in Human Cell Lines
- 2010
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 5:3, s. e9762-
-
Tidskriftsartikel (refereegranskat)abstract
- Several recent studies have indicated that transcription is pervasive in regions outside of protein coding genes and that short antisense transcripts can originate from the promoter and terminator regions of genes. Here we investigate transcription of fragments longer than 200 nucleotides, focusing on antisense transcription for known protein coding genes and intergenic transcription. We find that roughly 12% to 16% of all reads that originate from promoter and terminator regions, respectively, map antisense to the gene in question. Furthermore, we detect a high number of novel transcriptionally active regions (TARs) that are generally expressed at a lower level than protein coding genes. We find that the correlation between RNA-seq data and microarray data is dependent on the gene length, with longer genes showing a better correlation. We detect high antisense transcriptional activity from promoter, terminator and intron regions of protein-coding genes and identify a vast number of previously unidentified TARs, including putative novel EGFR transcripts. This shows that in-depth analysis of the transcriptome using RNA-seq is a valuable tool for understanding complex transcriptional events. Furthermore, the development of new algorithms for estimation of gene expression from RNA-seq data is necessary to minimize length bias.
|
|
| 10. |
- Klevebring, Daniel, 1981-
(författare)
-
On Transcriptome Sequencing
- 2009
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis is about the use of massive DNA sequencing to investigate the transcriptome. During recent decades, several studies have made it clear that the transcriptome comprises a more complex set of biochemical machinery than was previously believed. The majority of the genome can be expressed as transcripts; and overlapping and antisense transcription is widespread. New technologies for the interroga- tion of nucleic acids have made it possible to investigate such cellular phenomena in much greater detail than ever before. For each application, special requirements need to be met. The work presented in this thesis focuses on the transcrip- tome and the development of technology for its analysis. In paper I, we report our development of an automated approach for sample preparation. The procedure was benchmarked against a publicly available reference data set, and we note that our approach outperformed similar manual procedures in terms of reproducibility. In the work reported in papers II-IV, we used different massive sequencing technologies to investigate the transcriptome. In paper II we describe a concatemerization approach that increased throughput by 65% using 454 sequencing,and we identify classes of transcripts not previously described in Populus. Papers III and IV both report studies based on SOLiD sequencing. In the former, we investigated transcripts and proteins for 13% of the human gene and detected a massive overlap for the upper 50% transcriptional levels. In the work described in paper IV, we investigated transcription in non-genic regions of the genome and detected expression from a high number of previ- ously unknown loci.
|
|
