Magne Skårn - Academia.edu (original) (raw)

Papers by Magne Skårn

(A) Methylation-specific PCR (MSP) analyses of the CGIs, before and a... more (A) Methylation-specific PCR (MSP) analyses of the CGIs, before and after treatment with 5-Aza-2′-deoxycytidine (5-Aza). U and M, unmethylated and methylated products. Both standard gel images and 3D densitograms of the signal intensities are shown. (B) Schematic representation of the mir-142 locus. Locations of MSP amplicon #1 and #2 are indicated by arrows. 18 CpGs (region #1) and 14 CpGs (region #2) were subjected to bisulfite sequencing. CpG-containing transcription factor binding motifs are enclosed by boxes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079231#pone-0079231-g003" target="_blank">Figure 3B</a> for more information). E-box, enhancer box. Numbers indicate the position relative to pre-miR-142 (+1 to +87). (C) Bisulfite sequencing of DNA from MG-63 cells before and after treatment with 5-Aza for 72 hours, and untreated IOR/OS14 cells. The Wilcoxon signed rank test was used to test for statistical differences between treated and untreated MG-63 cells, as described in more details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079231#s2" target="_blank">Materials and Methods</a>. A P value ≤0.05 was considered as significant. (D) Bisulfite sequencing of immortalized bone marrow-derived stromal cells (iMSC#3) and primary osteoblasts. (E) Bisulfite sequencing of K562 leukemia- and peripheral blood progenitor cells. Black and white circles represent methylated and unmethylated CpGs, respectively, and each row represents a single clone. Grey circles, not determined. Ten clones were sequenced (n = 10), with the exception of MG-63 cells (region #1, n = 13; region #2, n = 12). (F) The 2,031 bp upstream region of pre-mir-142 was cloned into the promoter-less luciferase reporter construct pCpGL-basic and in vitro methylated with M.SssI (pCpGL/2031_M.SssI) or mock-methylated (pCpGL/2031_mock). All three constructs were individually transfected into U-2 OS cells along with a Renilla reporter construct. The luciferase activity was measured after 48 hours and calculated relative to that of pCpGL-basic (set to 1). Each histogram shows the average relative luciferase activity, and the error bars show the standard deviation of biological experiments (n≥5). The Wilcoxon signed rank test was used to test for statistical differences and the P values are shown above the histograms. A P value ≤0.05 was considered as significant.</p

BMC Genomics, 2022

Background Osteosarcoma is the most common primary malignant tumour of bone occurring in children... more Background Osteosarcoma is the most common primary malignant tumour of bone occurring in children and young adolescents and is characterised by complex genetic and epigenetic changes. The miRNA miR-486-5p has been shown to be downregulated in osteosarcoma and in cancer in general. Results To investigate if the mir-486 locus is epigenetically regulated, we integrated DNA methylation and miR-486-5p expression data using cohorts of osteosarcoma cell lines and patient samples. A CpG island in the promoter of the ANK1 host gene of mir-486 was shown to be highly methylated in osteosarcoma cell lines as determined by methylation-specific PCR and direct bisulfite sequencing. High methylation levels were seen for osteosarcoma patient samples, xenografts and cell lines based on quantitative methylation-specific PCR. 5-Aza-2′-deoxycytidine treatment of osteosarcoma cell lines caused induction of miR-486-5p and ANK1, indicating common epigenetic regulation in osteosarcoma cell lines. When overe...

Sarcoma, 2012

Liposarcoma cell lines representin vitromodels for studying disease mechanisms at the cellular le... more Liposarcoma cell lines representin vitromodels for studying disease mechanisms at the cellular level and for preclinical evaluation of novel drugs. To date there are a limited number of well-characterized models available. In this study, nine immortal liposarcoma cell lines were evaluated for tumor-forming ability, stem cell- and differentiation potential, and metastatic potential, with the aim to generate a well-characterized liposarcoma cell line panel. Detailed stem cell and differentiation marker analyses were also performed. Five of the liposarcoma cell lines were tumorigenic, forming tumors in mice. Interestingly, tumor-forming ability correlated with high proliferative capacityin vitro. All the cell lines underwent adipocytic differentiation, but the degree varied. Surprisingly, the expression of stem cell and differentiation markers did not correlate well with function. Overall, the panel contains cell lines suited forin vivoanalyses (LPS141, SA-4, T778, SW872, and LISA-2), ...

2971 Human sarcomas, malignant tumors of mesenchymal origin, show recurrent copy number alteratio... more 2971 Human sarcomas, malignant tumors of mesenchymal origin, show recurrent copy number alterations of specific chromosomal segments. In order to identify novel alterations and target genes for the copy number changes, we have constructed a genomic microarray with bacterial- and P1 artificial chromosome (BAC and PAC) clones covering the human genome at 1 Mb resolution, as well as the 1q12-q25 region at high resolution (tiling-path). We have used this genomic microarray for array comparative genomic hybridization (array CGH) of a panel of 170 human sarcomas. Malignant peripheral nerve sheath tumors (MPNSTs) arise sporadically or as part of the neurofibromatosis type 1 (NF1) or -2 (NF2) autosomal inherited disorders. We have analyzed seven MPNSTs, and correlated DNA copy number changes to patient outcome. A segment of 17q was gained in all patients who died of cancer. In order to identify candidate genes with increased expression in 17q, we have expression profiled six of the seven samples analysed by array CGH. Results show a number of known and novel genes with increased expression and functions relevant to cancer biology. In addition, seven known miRNAs are located in the differentially amplified area. Using Exiqon miRNA microarrays and Taqman assays we are investigating their expression. In addition, the impact of DNA copy number alterations will be correlated to mRNA and miRNA expression genome-wide. This and previous work shows the potential use of array CGH to distinguish patients with different clinical outcome, although further validation is required on larger tumor panels.

Transgenic Research, 2006

Transgene silencing in plants is most often dependent on homologous sequences, e.g. tandemly repe... more Transgene silencing in plants is most often dependent on homologous sequences, e.g. tandemly repeated T-DNAs. We have identified an Arabidopsis line (ex2-4 line 4) displaying silencing of the T-DNA-born nptII gene. This line contains a truncated copy of the T-DNA encompassing the nptII gene with its nos promoter adjacent to an intact T-DNA copy. The orientation of the intact and the truncated copies preclude the generation of a double-stranded nptII transcript. Therefore, we have investigated the genomic landscape surrounding T-DNA insertion in the silenced ex2-4 line 4 and five single-copy ex2-4 lines without silencing in search of features that might explain the silencing phenomenon. GC content, putative matrix-attachment regions and transcriptional interference from neighbouring genes could all be ruled out as major causes of silencing. Bisulphite sequencing revealed de novo methylation of the nos promoter both in non-silenced and silenced plants of this line, thus silencing was not correlated to DNA methylation level. Also, the methylation pattern deviated from that characteristic for RNA-mediated DNA methylation and silencing. Our data therefore suggest that ex2-4 line 4 represents a case where silencing is due to DNA-DNA pairing, i.e. pairing between the intact T-DNA and the adjacent truncated, inverted T-DNA copy.

Stem Cells and Development, 2012

PLoS ONE, 2012

Background: Osteosarcomas are the most common non-haematological primary malignant tumours of bon... more Background: Osteosarcomas are the most common non-haematological primary malignant tumours of bone, and all conventional osteosarcomas are high-grade tumours showing complex genomic aberrations. We have integrated genomewide genetic and epigenetic profiles from the EuroBoNeT panel of 19 human osteosarcoma cell lines based on microarray technologies.

Nucleic Acids Research, 2002

Molecular Cancer, 2008

Background Malignant peripheral nerve sheath tumors (MPNSTs) are rare and highly aggressive soft ... more Background Malignant peripheral nerve sheath tumors (MPNSTs) are rare and highly aggressive soft tissue tumors showing complex chromosomal aberrations. In order to identify recurrent chromosomal regions of gain and loss, and thereby novel gene targets of potential importance for MPNST development and/or progression, we have analyzed DNA copy number changes in seven high-grade MPNSTs using microarray-based comparative genomic hybridization (array CGH). Results Considerable more gains than losses were observed, and the most frequent minimal recurrent regions of gain included 1q24.1-q24.2, 1q24.3-q25.1, 8p23.1-p12, 9q34.11-q34.13 and 17q23.2-q25.3, all gained in five of seven samples. The 17q23.2-q25.3 region was gained in all five patients with poor outcome and not in the two patients with disease-free survival. cDNA microarray analysis and quantitative real-time reverse transcription PCR were used to investigate expression of genes located within these regions. The gene lysyl oxidase...

Cancer Research, 2012

Epigenetic and genetic alterations of microRNAs (miRNAs) are frequently seen in cancer, and are r... more Epigenetic and genetic alterations of microRNAs (miRNAs) are frequently seen in cancer, and are responsible for the deregulation of differentiation and proliferation programs. The levels of transcription of miRNAs have been directly linked to epigenetic silencing, i.e. hypermethylation of CpG islands in tumors. Osteosarcoma is the most common primary malignant bone tumor and shows complex genomic aberrations. Using Illumina9s Infinium technology we have profiled global promoter methylation of the EuroBoNeT osteosarcoma cell line panel (eurobonet.eu), in addition to a small panel of normal samples (osteoblast primary cultures and normal bone). A list of miRNAs that could be associated to a promoter CpG island was generated by identifying miRNAs that are encoded within transcripts of protein-coding host genes, or within 10 kb from a CpG island. Using an integrative approach, we have analyzed miRNA expression patterns together with DNA methylation data. Interestingly, hypermethylation of the upstream promoter seems to strongly suppress miRNA expression, and a high inverse correlation was observed between the degree of DNA methylation and expression of 17 miRNAs. The identified miRNAs were associated with hypermethylation in a high proportion of the osteosarcoma cell lines and hypomethylation in normal samples, suggesting that their transcription is regulated by promoter methylation, and that their silencing may play a role in osteosarcoma biology. To study this further, a subset of the cell lines was treated with a DNA demethylation agent. The relative expression levels of 8 miRNAs were found to increase upon treatment, as determined by quantitative RT-PCR. Thus, the transcription of these miRNAs seems to be regulated by epigenetic mechanisms. The methylation status of CpG islands associated with the most interesting miRNAs was then analyzed in more detail using methylation-specific PCR and direct bisulphite sequencing. We are currently expanding these investigations to a large tumor set where we will use quantitative methylation-specific PCR as a tool to determine the methylation status. Based on our findings, methylation of CpG islands seems to play a crucial role in controlling miRNA expression in osteosarcomas. This reveals new possibilities for activation of tumor-suppressing miRNAs by epigenetic treatment of osteosarcomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 197. doi:1538-7445.AM2012-197

Cancer Research, 2012

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Osteosarcomas are t... more Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Osteosarcomas are the most common primary malignant tumors of bone. The tumors are highly aggressive with poor prognosis, and display complex genomic aberrations. Previously, our group has identified a frequently deleted region in 3q13.31 in osteosarcoma clinical samples and cell lines [1]. The deleted region contains non-coding RNA genes, pseudogenes and the gene encoding the limbic-system associated membrane protein (LSAMP). The latter has previously been reported to be a candidate tumor suppressor gene in other cancer types, and more recently in osteosarcomas. Interestingly, our data show that low expression of LSAMP is statistically correlated with shorter patient survival. To examine the possible function of LSAMP in osteosarcomas, the expression was restored in an osteosarcoma cell line with a homozygous deletion of the gene. Characterization of the cell line with restored LSAMP expression showed decreased proliferation rate, which is consistent with its suggested role as a tumor suppressor gene. The migration rate and colony forming capability of the cells were unaffected. Interestingly, gene expression profiling of the restored lines, showed up-regulation of three genes, all proposed to have a role in cancer biology. The results indicate that the LSAMP protein might up-regulate the transcription of these three genes, and that LSAMP alone, or in conjunction with other genes, suppresses tumors by reducing their proliferation rate. Together, our data strengthens the hypothesis of LSAMP being a tumor suppressor gene in osteosarcomas. 1. Kresse, S.H., et al., LSAMP, a Novel Candidate Tumor Suppressor Gene in Human Osteosarcomas, Identified by Array Comparative Genomic Hybridization. Genes Chromosomes & Cancer, 2009. 48(8): p. 679-693. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4000. doi:1538-7445.AM2012-4000

BMC Genomics, 2022

Sarcoma, 2012

Transgenic Research, 2006

Stem Cells and Development, 2012

PLoS ONE, 2012

Nucleic Acids Research, 2002

Molecular Cancer, 2008

Cancer Research, 2012