Publications by authors named "Trond Stokke"

55 Publications

Karonudib has potent anti-tumor effects in preclinical models of B-cell lymphoma.

Sci Rep 2021 03 18;11(1):6317. Epub 2021 Mar 18.

Department of Cancer Immunology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Ullernschausseen 70, 0379, Montebello, Oslo, Norway.

Chemo-immunotherapy has improved survival in B-cell lymphoma patients, but refractory/relapsed diseases still represent a major challenge, urging for development of new therapeutics. Karonudib (TH1579) was developed to inhibit MTH1, an enzyme preventing oxidized dNTP-incorporation in DNA. MTH1 is highly upregulated in tumor biopsies from patients with diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma, hence confirming a rationale for targeting MTH1. Here, we tested the efficacy of karonudib in vitro and in preclinical B-cell lymphoma models. Using a range of B-cell lymphoma cell lines, karonudib strongly reduced viability at concentrations well tolerated by activated normal B cells. In B-cell lymphoma cells, karonudib increased incorporation of 8-oxo-dGTP into DNA, and prominently induced prometaphase arrest and apoptosis due to failure in spindle assembly. MTH1 knockout cell lines were less sensitive to karonudib-induced apoptosis, but were displaying cell cycle arrest phenotype similar to the wild type cells, indicating a dual inhibitory role of the drug. Karonudib was highly potent as single agent in two different lymphoma xenograft models, including an ABC DLBCL patient derived xenograft, leading to prolonged survival and fully controlled tumor growth. Together, our preclinical findings provide a rationale for further clinical testing of karonudib in B-cell lymphoma.
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http://dx.doi.org/10.1038/s41598-021-85613-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7973795PMC
March 2021

MRI Distinguishes Tumor Hypoxia Levels of Different Prognostic and Biological Significance in Cervical Cancer.

Cancer Res 2020 09 30;80(18):3993-4003. Epub 2020 Jun 30.

Department of Radiation Biology, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.

Tumor hypoxia levels range from mild to severe and have different biological and therapeutical consequences but are not easily assessable in patients. Here we present a method based on diagnostic dynamic contrast enhanced (DCE) MRI that reflects a continuous range of hypoxia levels in patients with tumors of cervical cancer. Hypoxia images were generated using an established approach based on pixel-wise combination of DCE-MRI parameters and , representing oxygen consumption and supply, respectively. Using two tumor models, an algorithm to retrieve surrogate measures of hypoxia levels from the images was developed and validated by comparing the MRI-defined levels with hypoxia levels reflected in pimonidazole-stained histologic sections. An additional indicator of hypoxia levels in patient tumors was established on the basis of expression of nine hypoxia-responsive genes; a strong correlation was found between these indicator values and MRI-defined hypoxia levels in 63 patients. Chemoradiotherapy outcome of 74 patients was most strongly predicted by moderate hypoxia levels, whereas more severe or milder levels were less predictive. By combining gene expression profiles and MRI-defined hypoxia levels in cancer hallmark analysis, we identified a distribution of levels associated with each hallmark; oxidative phosphorylation and G-M checkpoint were associated with moderate hypoxia, epithelial-to-mesenchymal transition, and inflammatory responses with significantly more severe levels. At the mildest levels, IFN response hallmarks together with HIF1A protein expression by IHC appeared significant. Thus, our method visualizes the distribution of hypoxia levels within patient tumors and has potential to distinguish levels of different prognostic and biological significance. SIGNIFICANCE: These findings present an approach to image a continuous range of hypoxia levels in tumors and demonstrate the combination of imaging with molecular data to better understand the biology behind these different levels.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0950DOI Listing
September 2020

Lu-Lilotomab Satetraxetan Has the Potential to Counteract Resistance to Rituximab in Non-Hodgkin Lymphoma.

J Nucl Med 2020 10 3;61(10):1468-1475. Epub 2020 Apr 3.

Nordic Nanovector ASA, Oslo, Norway.

Patients with non-Hodgkin lymphoma (NHL) who are treated with rituximab may develop resistant disease, often associated with changes in expression of CD20. The next-generation β-particle-emitting radioimmunoconjugate Lu-lilotomab-satetraxetan (Betalutin) was shown to up-regulate CD20 expression in different rituximab-sensitive NHL cell lines and to act synergistically with rituximab in a rituximab-sensitive NHL animal model. We hypothesized that Lu-lilotomab-satetraxetan may be used to reverse rituximab resistance in NHL. The rituximab-resistant Raji2R and the parental Raji cell lines were used. CD20 expression was measured by flow cytometry. Antibody-dependent cellular cytotoxicity (ADCC) was measured by a bioluminescence reporter assay. The efficacies of combined treatments with Lu-lilotomab-satetraxetan (150 or 350 MBq/kg) and rituximab (4 × 10 mg/kg) were compared with those of single agents or phosphate-buffered saline in a Raji2R-xenograft model. Cox regression and the Bliss independence model were used to assess synergism. Rituximab binding in Raji2R cells was 36% ± 5% of that in the rituximab-sensitive Raji cells. Lu-lilotomab-satetraxetan treatment of Raji2R cells increased the binding to 53% ± 3% of the parental cell line. Rituximab ADCC induction in Raji2R cells was 20% ± 2% of that induced in Raji cells, whereas treatment with Lu-lilotomab-satetraxetan increased the ADCC induction to 30% ± 3% of that in Raji cells, representing a 50% increase ( < 0.05). The combination of rituximab with 350 MBq/kg Lu-lilotomab-satetraxetan synergistically suppressed Raji2R tumor growth in athymic Foxn1 mice. Lu-lilotomab-satetraxetan has the potential to reverse rituximab resistance; it can increase rituximab binding and ADCC activity and can synergistically improve antitumor efficacy .
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http://dx.doi.org/10.2967/jnumed.119.237230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7539655PMC
October 2020

Selective Killing of Activated T Cells by 5-Aminolevulinic Acid Mediated Photodynamic Effect: Potential Improvement of Extracorporeal Photopheresis.

Cancers (Basel) 2020 Feb 6;12(2). Epub 2020 Feb 6.

Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, N-0379 Oslo, Norway.

Extracorporeal photopheresis (ECP), a modality that exposes isolated leukocytes to the photosensitizer 8-methoxypsoralen (8-MOP) and ultraviolet-A (UV-A) light, is used to treat conditions such as cutaneous T-cell lymphoma and graft-versus-host disease. However, the current procedure of ECP has limited selectivity and efficiency; and produces only partial response in the majority of treated patients. Additionally, the treatment is expensive and time-consuming, so the improvement for this modality is needed. In this study, we used the concept of photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA), a precursor of an endogenously synthesized photosensitizer protoporphyrin IX (PpIX) in combination with blue light to explore the possibility of targeting activated human blood T cells ex vivo. With various T-cell activation protocols, a high ALA-induced PpIX production took place in activated CD3, CD4CD25, and CD8 T cell populations with their subsequent killing after blue light exposure. By contrast, resting T cells were much less damaged by the treatment. The selective and effective killing effect on the activated cells was also seen after co-cultivating activated and resting T cells. Under our clinically relevant experimental conditions, ALA-PDT killed activated T cells more selectively and efficiently than 8-MOP/UV-A. Monocyte-derived dendritic cells (DCs) were not affected by the treatment. Incubation of ALA-PDT damaged T cells with autologous DCs induced a downregulation of the co-stimulatory molecules CD80/CD86 and also upregulation of interleukin 10 (IL-10) and indoleamine 2,3-dioxygenase expression, two immunosuppressive factors that may account for the generation of tolerogenic DCs. Overall, the data support the potential use of ALA-PDT strategy for improving ECP by selective and effective killing of activated T cells and induction of immune tolerance.
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http://dx.doi.org/10.3390/cancers12020377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072277PMC
February 2020

Cell Cycle Analysis and Relevance for Single-Cell Gating in Mass Cytometry.

Cytometry A 2020 08 14;97(8):832-844. Epub 2020 Jan 14.

Department of Core Facilities, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway.

Cell cycle analysis by mass cytometry (MC) is hampered by the poor resolution of the Iridium-labeled DNA intercalator compared to DNA-specific fluorescent dyes. We report here a minimum cell cycle panel for MC consisting of Ir-intercalator (DNA content), IdU (S phase), anti-pS28HistoneH3 (mitosis), anti-CDT1 (G phase) and anti-Geminin (non-G phases). Cell cycle distributions obtained by MC were not significantly different from fluorescence flow cytometry results (r = 0.98, P < 0.001). Further subdivision of the G and G phases could be done with anti-pS780RB1 (late G ) and anti-PLK1 (late G ), respectively. A disadvantage of MC is that aggregates of cells cannot easily be removed while retaining all single cells. We have developed an analysis pipeline including unsupervised clustering by FlowSOM and subsequent single-cell gating. When performed on cells stained with the cell cycle panel, this analysis pipeline successfully identified debris, dead/apoptotic cells, nonsingle-cell populations and the major cell cycle phases. The presented cell cycle panel and analysis pipeline thus achieves true single-cell analysis at the same time as any additional channels in the panel are open for phenotyping and cell cycle-resolved expression or modification analysis. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.
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http://dx.doi.org/10.1002/cyto.a.23960DOI Listing
August 2020

A CEP104-CSPP1 Complex Is Required for Formation of Primary Cilia Competent in Hedgehog Signaling.

Cell Rep 2019 08;28(7):1907-1922.e6

Department of Radiation Biology, Institute for Cancer Research, OUH-Norwegian Radium Hospital, Oslo, Norway. Electronic address:

CEP104 is an evolutionarily conserved centrosomal and ciliary tip protein. CEP104 loss-of-function mutations are reported in patients with Joubert syndrome, but their function in the etiology of ciliopathies is poorly understood. Here, we show that cep104 silencing in zebrafish causes cilia-related manifestations: shortened cilia in Kupffer's vesicle, heart laterality, and cranial nerve development defects. We show that another Joubert syndrome-associated cilia tip protein, CSPP1, interacts with CEP104 at microtubules for the regulation of axoneme length. We demonstrate in human telomerase reverse transcriptase-immortalized retinal pigmented epithelium (hTERT-RPE1) cells that ciliary translocation of Smoothened in response to Hedgehog pathway stimulation is both CEP104 and CSPP1 dependent. However, CEP104 is not required for the ciliary recruitment of CSPP1, indicating that an intra-ciliary CEP104-CSPP1 complex controls axoneme length and Hedgehog signaling competence. Our in vivo and in vitro analyses of CEP104 define its interaction with CSPP1 as a requirement for the formation of Hedgehog signaling-competent cilia, defects that underlie Joubert syndrome.
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http://dx.doi.org/10.1016/j.celrep.2019.07.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702141PMC
August 2019

Mitochondrial Function of CKS2 Oncoprotein Links Oxidative Phosphorylation with Cell Division in Chemoradioresistant Cervical Cancer.

Neoplasia 2019 04 8;21(4):353-362. Epub 2019 Mar 8.

Department of Radiation Biology, Oslo University Hospital, Oslo, Norway. Electronic address:

CDK regulatory subunit 2 (CKS2) has a nuclear function that promotes cell division and is a candidate biomarker of chemoradioresistance in cervical cancer. The underlying mechanisms are, however, not completely understood. We investigated whether CKS2 also has a mitochondrial function that augments tumor aggressiveness. Based on global gene expression data of two cervical cancer cohorts of 150 and 135 patients, we identified a set of genes correlated with CKS2 expression. Gene set enrichment analysis showed enrichment of mitochondrial cellular compartments, and the hallmarks oxidative phosphorylation (OXPHOS) and targets of the MYC oncogene in the gene set. By in situ proximity ligation assay, we showed that CKS2 formed complex with the positively correlated MYC target, mitochondrial single-stranded DNA binding protein SSBP1, in the mitochondrion of cervix tumor samples and HeLa and SiHa cervical cancer cell lines, indicating a role in mitochondrial DNA (mtDNA) replication and thereby OXPHOS. CDK1 was found to be part of the complex. Flow cytometry analyses of HeLa cells showed cell cycle regulation of the CKS2-SSBP1 complex consistent with mtDNA replication activity. Moreover, repression of mtDNA replication and OXPHOS by acute hypoxia decreased CKS2-SSBP1 complex abundance and expression of MYC targets. By immunohistochemistry, cytoplasmic CKS2 expression was found to add to the prognostic impact of nuclear CKS2 expression in patients, suggesting that the mitochondrial function promotes tumor aggressiveness. Our study uncovers a novel link between regulation of cell division by nuclear pathways and OXPHOS in the mitochondrion that involves CKS2 and promotes chemoradioresistance of cervical cancer.
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http://dx.doi.org/10.1016/j.neo.2019.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411633PMC
April 2019

Combination of Lu-lilotomab with rituximab significantly improves the therapeutic outcome in preclinical models of non-Hodgkin's lymphoma.

Eur J Haematol 2018 Oct 31;101(4):522-531. Epub 2018 Aug 31.

Nordic Nanovector ASA, Oslo, Norway.

Objectives: To investigate the therapeutic potential of the next-generation anti-CD37 radioimmunoconjugate Lu-lilotomab satetraxetan ( Lu-lilotomab) in combination with the anti-CD20 antibody rituximab for treatment of mice with non-Hodgkin's lymphoma (NHL) xenografts.

Methods: Nude mice with subcutaneous (s.c.) Burkitt's lymphoma Daudi xenografts and SCID mice intravenously (i.v.) injected with Mantle cell lymphoma Rec-1 cells were treated with either Lu-lilotomab or rituximab alone or with the combination of both treatments. Tumour volume, body weight, blood counts and clinical status were monitored. CD20 expression was measured using flow cytometry with fluorescence-labelled rituximab.

Results: The combination of Lu-lilotomab and rituximab was synergistic for treatment of nude mice with s.c. Daudi xenografts while it was additive for treatment of SCID mice with i.v. injected Rec-1 cells. Binding of rituximab to NHL cells in-vitro was increased by pretreatment with Lu-lilotomab.

Conclusions: Treatment of mice with NHL xenografts with Lu-lilotomab synergistically increased tumour suppression of subsequent anti-CD20 immunotherapy and improved survival. If the same effect is confirmed in a recently started clinical study, it could change the way radioimmunotherapy and CD20 immunotherapy would be used in the future.
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http://dx.doi.org/10.1111/ejh.13139DOI Listing
October 2018

The E3 ubiquitin ligase UBR5 regulates centriolar satellite stability and primary cilia.

Mol Biol Cell 2018 07 9;29(13):1542-1554. Epub 2018 May 9.

Faculty of Medicine, School of Medical Sciences, University of New South Wales, Sydney 2052, Australia.

Primary cilia are crucial for signal transduction in a variety of pathways, including hedgehog and Wnt. Disruption of primary cilia formation (ciliogenesis) is linked to numerous developmental disorders (known as ciliopathies) and diseases, including cancer. The ubiquitin-proteasome system (UPS) component UBR5 was previously identified as a putative positive regulator of ciliogenesis in a functional genomics screen. UBR5 is an E3 ubiquitin ligase that is frequently deregulated in tumors, but its biological role in cancer is largely uncharacterized, partly due to a lack of understanding of interacting proteins and pathways. We validated the effect of UBR5 depletion on primary cilia formation using a robust model of ciliogenesis, and identified CSPP1, a centrosomal and ciliary protein required for cilia formation, as a UBR5-interacting protein. We show that UBR5 ubiquitylates CSPP1, and that UBR5 is required for cytoplasmic organization of CSPP1-comprising centriolar satellites in centrosomal periphery, suggesting that UBR5-mediated ubiquitylation of CSPP1 or associated centriolar satellite constituents is one underlying requirement for cilia expression. Hence, we have established a key role for UBR5 in ciliogenesis that may have important implications in understanding cancer pathophysiology.
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http://dx.doi.org/10.1091/mbc.E17-04-0248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6080653PMC
July 2018

Combined inhibition of Wee1 and Chk1 gives synergistic DNA damage in S-phase due to distinct regulation of CDK activity and CDC45 loading.

Oncotarget 2017 Feb;8(7):10966-10979

Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, N-0310, Norway.

Recent studies have shown synergistic cytotoxic effects of simultaneous Chk1- and Wee1-inhibition. However, the mechanisms behind this synergy are not known. Here, we present a flow cytometry-based screen for compounds that cause increased DNA damage in S-phase when combined with the Wee1-inhibitor MK1775. Strikingly, the Chk1-inhibitors AZD7762 and LY2603618 were among the top candidate hits of 1664 tested compounds, suggesting that the synergistic cytotoxic effects are due to increased S-phase DNA damage. Combined Wee1- and Chk1-inhibition caused a strong synergy in induction of S-phase DNA damage and reduction of clonogenic survival. To address the underlying mechanisms, we developed a novel assay measuring CDK-dependent phosphorylations in single S-phase cells. Surprisingly, while Wee1-inhibition alone induced less DNA damage compared to Chk1-inhibition, Wee1-inhibition caused a bigger increase in S-phase CDK-activity. However, the loading of replication initiation factor CDC45 was more increased after Chk1- than Wee1-inhibition and further increased by the combined treatment, and thus correlated well with DNA damage. Therefore, when Wee1 alone is inhibited, Chk1 suppresses CDC45 loading and thereby limits the extent of unscheduled replication initiation and subsequent S-phase DNA damage, despite very high CDK-activity. These results can explain why combined treatment with Wee1- and Chk1-inhibitors gives synergistic anti-cancer effects.
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http://dx.doi.org/10.18632/oncotarget.14089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5355238PMC
February 2017

Hypoxia-independent gene expression signature associated with radiosensitisation of prostate cancer cell lines by histone deacetylase inhibition.

Br J Cancer 2016 10 6;115(8):929-939. Epub 2016 Sep 6.

Department of Radiation Biology, Norwegian Radium Hospital, Oslo University Hospital, Pb 4950, Nydalen, 0424 Oslo, Norway.

Background: Histone deacetylase inhibitors (HDACis) like vorinostat are promising radiosensitisers in prostate cancer, but their effect under hypoxia is not known. We investigated gene expression associated with radiosensitisation of normoxic and hypoxic prostate cancer cells by vorinostat.

Methods: Cells were exposed to vorinostat under normoxia or hypoxia and subjected to gene expression profiling before irradiation and clonogenic survival analysis.

Results: Pretreatment with vorinostat led to radiosensitisation of the intrinsically radioresistant DU 145 cells, but not the radiosensitive PC-3 and 22Rv1 cells, and was independent of hypoxia status. Knockdown experiments showed that the sensitisation was not caused by repression of hypoxia-inducible factor HIF1 or tumour protein TP53. Global deregulation of DNA repair and chromatin organisation genes was associated with radiosensitisation under both normoxia and hypoxia. A radiosensitisation signature with expression changes of 56 genes was generated and valid for both conditions. For eight signature genes, baseline expression also correlated with sensitisation, showing potential as pretreatment biomarker. The hypoxia independence of the signature was confirmed in a clinical data set.

Conclusions: Pretreatment with HDACi may overcome radioresistance of hypoxic prostate tumours by similar mechanisms as under normoxia. We propose a gene signature to predict radiosensitising effects independent of hypoxia status.
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http://dx.doi.org/10.1038/bjc.2016.278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5061908PMC
October 2016

Analyzing Schizosaccharomyces pombe DNA Content by Flow Cytometry.

Cold Spring Harb Protoc 2016 06 1;2016(6). Epub 2016 Jun 1.

Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, Montebello, 0310 Oslo, Norway.

Flow cytometry can be used to measure the DNA content of individual cells. The data are usually presented as DNA histograms that can be used to examine the cells' progression through the cell cycle. Under standard growth conditions, fission yeast cells do not complete cytokinesis until after G1 phase; therefore, DNA histograms show one major peak representing cells in G1 (2×1C DNA) and G2 phase (1×2C DNA). By analysis of the duration of the fluorescence signal as well as the intensity of the DNA-related signal, it is possible to discriminate between cells in M/G1, S, and G2 This protocol describes how to prepare cells for flow cytometry and analyze them. We also describe the application of barcoding for more accurate comparison of samples.
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http://dx.doi.org/10.1101/pdb.prot091280DOI Listing
June 2016

New distinct compartments in the G2 phase of the cell cycle defined by the levels of γH2AX.

Cell Cycle 2015 ;14(20):3261-9

a Group for Molecular Radiation Biology; Department of Radiation Biology; The Norwegian Radium Hospital ; Oslo , Norway.

Induction of DNA double strand breaks leads to phosphorylation and focus-formation of H2AX. However, foci of phosphorylated H2AX (γH2AX) appear during DNA replication also in the absence of exogenously applied injury. We measured the amount and the number of foci of γH2AX in different phases of the cell cycle by flow cytometry, sorting and microscopy in 4 malignant B-lymphocyte cell lines. There were no detectable γH2AX and no γH2AX-foci in G1 cells in exponentially growing cells and cells treated with PARP inhibitor (PARPi) for 24 h to create damage and reduce DNA repair. The amount of γH2AX increased immediately upon S phase entry, and about 10 and 30 γH2AX foci were found in mid-S phase control and PARPi-treated cells, respectively. The γH2AX-labeled damage caused by DNA replication was not fully repaired before entry into G2. Intriguingly, G2 cells populated a continuous distribution of γH2AX levels, from cells with a high content of γH2AX and the same number of foci as S phase cells (termed "G2H" compartment), to cells that there were almost negative and had about 2 foci (termed "G2L" compartment). EdU-labeling of S phase cells revealed that G2H was directly populated from S phase, while G2L was populated from G2H, but in control cells also directly from S phase. The length of G2H in particular increased after PARPi treatment, compatible with longer DNA-repair times. Our results show that cells repair replication-induced damage in G2H, and enter mitosis after a 2-3 h delay in G2L.
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http://dx.doi.org/10.1080/15384101.2015.1087617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4827887PMC
August 2016

Replication-induced DNA damage after PARP inhibition causes G2 delay, and cell line-dependent apoptosis, necrosis and multinucleation.

Cell Cycle 2015 ;14(20):3248-60

a Group for Molecular Radiation Biology ; Department of Radiation Biology ; The Norwegian Radium Hospital ; Oslo , Norway.

PARP inhibitors have been approved for treatment of tumors with mutations in or loss of BRCA1/2. The molecular mechanisms and particularly the cellular phenotypes resulting in synthetic lethality are not well understood and varying clinical responses have been observed. We have investigated the dose- and time-dependency of cell growth, cell death and cell cycle traverse of 4 malignant lymphocyte cell lines treated with the PARP inhibitor Olaparib. PARP inhibition induced a severe growth inhibition in this cell line panel and increased the levels of phosphorylated H2AX-associated DNA damage in S phase. Repair of the remaining replication related damage caused a G2 phase delay before entry into mitosis. The G2 delay, and the growth inhibition, was more pronounced in the absence of functional ATM. Further, Olaparib treated Reh and Granta-519 cells died by apoptosis, while U698 and JVM-2 cells proceeded through mitosis with aberrant chromosomes, skipped cytokinesis, and eventually died by necrosis. The TP53-deficient U698 cells went through several rounds of DNA replication and mitosis without cytokinesis, ending up as multinucleated cells with DNA contents of up to 16c before dying. In summary, we report here for the first time cell cycle-resolved DNA damage induction, and cell line-dependent differences in the mode of cell death caused by PARP inhibition.
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http://dx.doi.org/10.1080/15384101.2015.1085137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4825575PMC
August 2016

CSPP-L Associates with the Desmosome of Polarized Epithelial Cells and Is Required for Normal Spheroid Formation.

PLoS One 2015 4;10(8):e0134789. Epub 2015 Aug 4.

Department of Radiation Biology, Division of Cancer Medicine, Surgery and Transplantation, Institute for Cancer Research, Oslo University Hospitals-Norwegian Radium Hospital, Oslo, Norway.

Deleterious mutations of the Centrosome/Spindle Pole associated Protein 1 gene, CSPP1, are causative for Joubert-syndrome and Joubert-related developmental disorders. These disorders are defined by a characteristic mal-development of the brain, but frequently involve renal and hepatic cyst formation. CSPP-L, the large protein isoform of CSPP1 localizes to microtubule ends of the mitotic mid-spindle and the ciliary axoneme, and is required for ciliogenesis. We here report the microtubule independent but Desmoplakin dependent localization of CSPP-L to Desmosomes in apical-basal polarized epithelial cells. Importantly, siRNA conferred depletion of CSPP-L or Desmoplakin promoted multi-lumen spheroid formation in 3D-cultures of non-ciliated human colon carcinoma Caco-2 cells. Multi-lumen spheroids of CSPP1 siRNA transfectants showed disrupted apical cell junction localization of the cytoskeleton organizing RhoGEF ECT2. Our results hence identify a novel, non-ciliary role for CSPP-L in epithelial morphogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0134789PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4524657PMC
May 2016

Mitochondrial DNA Deficiency in Ovarian Cancer Cells and Cancer Stem Cell-like Properties.

Anticancer Res 2015 Jul;35(7):3743-53

Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway Department of Pathology, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway

Background: A low quantity of mitochondrial DNA (mtDNA) is a risk factor in a variety of tumor types. However, it is unclear how mtDNA reduction influences tumor behavior.

Material And Methods: mtDNA-deficient ovarian cancer cells were established by ethidium bromide (EtBr) treatment with additive combination of pyruvate and uridine.

Results: The mtDNA-deficient cells had a low growth and colony-forming efficiency compared to the control cells. RNA sequencing revealed down-regulation of mitochondrion-related genes and up-regulation of genes related to cell proliferation and anti-apoptosis. The expression of genes involved in cancer metastasis, proliferation, angiogenesis, drug resistance and cancer cell stemness were also up-regulated. Intriguingly, cancer stem cell markers CD90 and CD117 were both up-regulated by EtBr dose-dependently in both cell lines.

Conclusion: MtDNA deficiency may induce ovarian cancer stem cell-like properties through different ways in vitro, therefore contributing to different tumor behaviors.
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July 2015

Karyotyping of diffuse large B-cell lymphomas: loss of 17p is associated with poor patient outcome.

Eur J Haematol 2013 Oct 17;91(4):332-8. Epub 2013 Aug 17.

Department of Immunology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway.

Background: Cytogenetic studies of patients with diffuse large B-cell lymphoma (DLBCL) have revealed a large spectrum of chromosomal abnormalities, some of which may be clinically relevant. We wanted to evaluate possible associations between commonly acquired chromosome aberrations and prognosis in a large cohort of patients.

Methods: All patients with DLBCL treated at our center during 1999-2010 with an abnormal G-banding karyotype determined on cells short-term cultured from diagnostic biopsies were included. Detailed information on staging, treatment, and outcome was available for all patients.

Results: Of the 110 patients available for analysis, there were 48 deaths and 27 relapses after a median follow-up of 4.5 yr. Eleven different chromosomal abnormalities were detected in more than ten percent of patients. Of those, only loss of 17p, including the TP53 tumor suppressor gene, was significantly associated with inferior long-term prognosis. Five year overall and progression-free survival frequencies were 32% and 27% for patients with loss of 17p and 67% and 59% in patients without this abnormality.

Conclusion: In a relatively large cohort of patients with DLBCL analyzed by chromosome banding, loss of 17p was the only chromosomal abnormality associated with inferior survival in uni- and multivariate analysis.
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http://dx.doi.org/10.1111/ejh.12171DOI Listing
October 2013

Identification of eight candidate target genes of the recurrent 3p12-p14 loss in cervical cancer by integrative genomic profiling.

J Pathol 2013 May 14;230(1):59-69. Epub 2013 Mar 14.

Department of Radiation Biology, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.

The pathogenetic role, including its target genes, of the recurrent 3p12-p14 loss in cervical cancer has remained unclear. To determine the onset of the event during carcinogenesis, we used microarray techniques and found that the loss was the most frequent 3p event, occurring in 61% of 92 invasive carcinomas, in only 2% of 43 high-grade intraepithelial lesions (CIN2/3), and in 33% of 6 CIN3 lesions adjacent to invasive carcinomas, suggesting a role in acquisition of invasiveness or early during the invasive phase. We performed an integrative DNA copy number and expression analysis of 77 invasive carcinomas, where all genes within the recurrent region were included. We selected eight genes, THOC7, PSMD6, SLC25A26, TMF1, RYBP, SHQ1, EBLN2, and GBE1, which were highly down-regulated in cases with loss, as confirmed at the protein level for RYBP and TMF1 by immunohistochemistry. The eight genes were subjected to network analysis based on the expression profiles, revealing interaction partners of proteins encoded by the genes that were coordinately regulated in tumours with loss. Several partners were shared among the eight genes, indicating crosstalk in their signalling. Gene ontology analysis showed enrichment of biological processes such as apoptosis, proliferation, and stress response in the network and suggested a relationship between down-regulation of the eight genes and activation of tumourigenic pathways. Survival analysis showed prognostic impact of the eight-gene signature that was confirmed in a validation cohort of 74 patients and was independent of clinical parameters. These results support the role of the eight candidate genes as targets of the 3p12-p14 loss in cervical cancer and suggest that the strong selection advantage of the loss during carcinogenesis might be caused by a synergetic effect of several tumourigenic processes controlled by these targets.
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http://dx.doi.org/10.1002/path.4168DOI Listing
May 2013

Prostate cancer cell lines under hypoxia exhibit greater stem-like properties.

PLoS One 2011 28;6(12):e29170. Epub 2011 Dec 28.

Department of Pathology, University of Oslo, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.

Hypoxia is an important environmental change in many cancers. Hypoxic niches can be occupied by cancer stem/progenitor-like cells that are associated with tumor progression and resistance to radiotherapy and chemotherapy. However, it has not yet been fully elucidated how hypoxia influences the stem-like properties of prostate cancer cells. In this report, we investigated the effects of hypoxia on human prostate cancer cell lines, PC-3 and DU145. In comparison to normoxia (20% O(2)), 7% O(2) induced higher expressions of HIF-1α and HIF-2α, which were associated with upregulation of Oct3/4 and Nanog; 1% O(2) induced even greater levels of these factors. The upregulated NANOG mRNA expression in hypoxia was confirmed to be predominantly retrogene NANOGP8. Similar growth rates were observed for cells cultivated under hypoxic and normoxic conditions for 48 hours; however, the colony formation assay revealed that 48 hours of hypoxic pretreatment resulted in the formation of more colonies. Treatment with 1% O(2) also extended the G(0)/G(1) stage, resulting in more side population cells, and induced CD44 and ABCG2 expressions. Hypoxia also increased the number of cells positive for ABCG2 expression, which were predominantly found to be CD44(bright) cells. Correspondingly, the sorted CD44(bright) cells expressed higher levels of ABCG2, Oct3/4, and Nanog than CD44(dim) cells, and hypoxic pretreatment significantly increased the expressions of these factors. CD44(bright) cells under normoxia formed significantly more colonies and spheres compared with the CD44(dim) cells, and hypoxic pretreatment even increased this effect. Our data indicate that prostate cancer cells under hypoxia possess greater stem-like properties.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0029170PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3247249PMC
May 2012

Bone morphogenetic proteins inhibit CD40L/IL-21-induced Ig production in human B cells: differential effects of BMP-6 and BMP-7.

Eur J Immunol 2011 Nov 18;41(11):3135-45. Epub 2011 Oct 18.

Department of Immunology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway.

Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily. TGF-β can affect class switch recombination in human B cells, but whether BMPs also play a role have not been tested. We investigated the functional effects of exogenously added BMPs on CD27(-) naive and CD27(+) memory B cells from healthy donors. BMP-2, -4, -6 and -7 inhibited CD40L/IL-21-induced production of IgM, IgG and IgA. BMP-6 reduced Ig production by 70% in memory B cells and more than 55% in naive B cells, whereas the other BMPs were slightly less potent. We observed a striking difference in functional effects between the structurally similar BMP-6 and BMP-7, as BMP-6 mainly inhibited plasmablast differentiation, and BMP-7 mainly induced apoptosis. In memory B cells, BMP-6 upregulated expression of DNA-binding protein inhibitor genes, but potently inhibited CD40L/IL-21-induced upregulation of the transcription factor XBP1, necessary for the late stages of plasmacytic differentiation. Expression of transcription factors regulating earlier stages (IRF4, PRDM1) was not affected by BMP-6. Taken together, these results show that BMPs are potent suppressors of naive and memory B cells.
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http://dx.doi.org/10.1002/eji.201141558DOI Listing
November 2011

Side population cells in highly enriched CD34-positive cells from peripheral blood progenitor cells identify an immature subtype of hematopoietic progenitor cells but do not predict time to engraftment in patients treated with high-dose therapy.

Eur J Haematol 2011 Dec 15;87(6):494-502. Epub 2011 Sep 15.

Departments of Cellular Therapy Immunology Oncology Biophysics, The Norwegian Radiumhospital, Oslo University Hospital, Norway.

Objective: A Hoechst 33342 dye efflux assay can be used to define a population of immature hematopoietic progenitor cells (HPC) that are called side population (SP) cells. Previously, SP cells examined from bone marrow (BM) and peripheral blood progenitor cells (PBPC) were found to be predominantly CD34 negative.

Methods And Results: In this study, we show that the level of CD34+ cells within the SP fraction increases from 2% in BM to 15% in mobilized PBPC. Furthermore, SP cells are found in highly enriched CD34+ cells from both BM and PBPC, and these cells define an immature phenotype of HPC. We also observed a higher level of CD133+ cells within the SPCD34+ cell population. Moreover, the frequency of long-term culture-initiating cells (LTC-IC) was markedly increased in SPCD34+ cells. To further investigate whether variations in the level of SP cells in the CD34+ cell fraction influenced short-term engraftment, we studied 20 patients with Hodgkin lymphoma that were autotransplanted with highly enriched CD34+ cells from PB. The percentage of SP cells in the PBCD34+ cell fraction was highly variable, ranging from 0.3 to 22%. No correlation was found between the content of SP cells in the autotransplanted CD34+ cells and time to short-term engraftment.

Conclusion: SPCD34+ cells in PBPC define an immature phenotype of HPC with increased numbers of LTC-IC, and they are more frequently found in PBPC than in BM. The number of SP cells does not predict time to engraftment.
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http://dx.doi.org/10.1111/j.1600-0609.2011.01681.xDOI Listing
December 2011

The amount of DNA damage needed to activate the radiation-induced G2 checkpoint varies between single cells.

Radiother Oncol 2011 Oct 30;101(1):24-7. Epub 2011 Jun 30.

Department of Radiation Biology, Oslo University Hospital, Norway.

Background And Purpose: The radiation-induced G2 checkpoint helps facilitate DNA repair before cell division. However, recent work has revealed that human cells often escape the G2 checkpoint with unrepaired DNA breaks. The purpose was to explore whether G2 checkpoint activation occurs according to a threshold level of DNA damage.

Materials And Methods: G2 checkpoint activation was assayed at 75-90 min and 24-48 h after X-ray irradiation of BJ diploid fibroblasts and U2OS osteosarcoma cells. Multiparameter flow cytometry with pacific blue barcoding, and flow cytometry-based sorting of phospho-H3 positive cells to microscope slides, were used to examine the DNA damage marker γ-H2AX in individual mitotic cells that had escaped the G2 checkpoint.

Results: For all radiation doses and times tested, the number of γ-H2AX foci varied between individual mitotic cells. At 75 min the median levels of γ-H2AX in mitotic cells increased with higher radiation doses. At 24-48 h, following a prolonged G2 checkpoint, cells were more resistant to checkpoint re-activation by a second dose of radiation.

Conclusion: Our results suggest that different amounts of DNA damage are needed to activate the G2 checkpoint in individual cells. Such single cell variation in checkpoint activation may potentially contribute to radiation-induced genomic instability.
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http://dx.doi.org/10.1016/j.radonc.2011.05.060DOI Listing
October 2011

Cell-cycle analysis of fission yeast cells by flow cytometry.

PLoS One 2011 Feb 28;6(2):e17175. Epub 2011 Feb 28.

Department of Cell Biology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway.

The cell cycle of the fission yeast, Schizosaccharomyces pombe, does not easily lend itself to analysis by flow cytometry, mainly because cells in G(1) and G(2) phase contain the same amount of DNA. This occurs because fission yeast cells under standard growth conditions do not complete cytokinesis until after G(1) phase. We have devised a flow cytometric method exploiting the fact that cells in G(1) phase contain two nuclei, whereas cells in G(2) are mononuclear. Measurements of the width as well as the total area of the DNA-associated fluorescence signal allows the discrimination between cells in G(1) and in G(2) phase and the cell-cycle progression of fission yeast can be followed in detail by flow cytometry. Furthermore, we show how this method can be used to monitor the timing of cell entry into anaphase. Fission yeast cells tend to form multimers, which represents another problem of flow cytometry-based cell-cycle analysis. Here we present a method employing light-scatter measurements to enable the exclusion of cell doublets, thereby further improving the analysis of fission yeast cells by flow cytometry.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0017175PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3046126PMC
February 2011

Three independent mechanisms for arrest in G2 after ionizing radiation.

Cell Cycle 2011 Mar 1;10(5):819-29. Epub 2011 Mar 1.

Department of Radiation Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo, Norway.

Cell cycle checkpoints ensure that eukaryotic cells do not enter mitosis after ionizing irradiation (IR). The G(2)-arrest after IR is the result of activation of multiple signalling pathways, the contributions of which vary with time after irradiation. We have studied the time evolution of the IR-induced G(2)-arrest in human B-lymphocyte cancer cell lines, as well as the molecular mechanisms responsible for the arrest. Cells that were in G(2) phase at the time of irradiation experienced a transient arrest that blocked entry into mitosis at 0-2 hours after IR (0.5 or 4 Gy). Activation of ATM and CHEK2 occurred at the same time as this early arrest and was, like the arrest, abrogated by the ATM-inhibitor KU-55933. A late, permanent and ATM-independent arrest (≥6 hours after IR) of cells that were in G(2)/S/G(1) at the time of irradiation (4 Gy) was inactivated by caffeine. This late G(2)-arrest could not be explained by down-regulation of genes with functions in G(2)/mitosis (e.g. PLK1, CCNB1/2), since the down-regulation was transient and not accompanied by reduced protein levels. However, the persistent phosphorylation of CHEK1 after 4 Gy suggested a role for CHEK1 in the late arrest, consistent with the abrogation of the arrest in CHEK1-depleted cells. TP53 was not necessary for the late G(2)-arrest, but mediated an intermediate arrest (2-10 hours after IR) independently of ATM and CHEK1. In conclusion, the IR-induced arrest in G(2) is mediated by ATM immediately after irradiation, with TP53 for independent and transient back-up, while CHEK1 is necessary for the late arrest.
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http://dx.doi.org/10.4161/cc.10.5.14968DOI Listing
March 2011

CSPP is a ciliary protein interacting with Nephrocystin 8 and required for cilia formation.

Mol Biol Cell 2010 Aug 2;21(15):2555-67. Epub 2010 Jun 2.

Department of Radiation Biology, Institute for Cancer Research, The Norwegian Radium Hospital, N-0310 Oslo, Norway.

We described previously the cell cycle- and microtubule-related functions of two splice isoforms of the centrosome spindle pole-associated protein (CSPP and CSPP-L). Here, we show that endogenous CSPP isoforms not only localize to centrosomes and the midbody in cycling cells but also extend to the cilia axoneme in postmitotic resting cells. They are required for ciliogenesis in hTERT-RPE1 cells in vitro and are expressed in ciliated renal, retinal, and respiratory cells in vivo. We report that CSPP isoforms require their common C-terminal domain to interact with Nephrocystin 8 (NPHP8/RPGRIP1L) and to form a ternary complex with NPHP8 and NPHP4. We find CSPP-L to be required for the efficient localization of NPHP8 but not NPHP4 to the basal body. The ciliogenesis defect in hTERT-RPE1 cells is, however, not mediated through loss of NPHP8. Similar to the effects of ectopical expression of CSPP-L, cilia length increased in NPHP8-depleted cells. Our results thus suggest that CSPP proteins may be involved in further cytoskeletal organization of the basal body and its primary cilium. To conclude, we have identified a novel, nonmitotic function of CSPP proteins placing them into a ciliary protein network crucial for normal renal and retinal tissue architecture and physiology.
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http://dx.doi.org/10.1091/mbc.e09-06-0503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2912343PMC
August 2010

Mechanisms of the elimination of low dose hyper-radiosensitivity in T-47D cells by low dose-rate priming.

Int J Radiat Biol 2009 Dec;85(12):1157-65

Department of Physics, University of Oslo, Biophysics Group, PB 1048 Blindern, N-0316 Oslo, Norway.

Purpose: To investigate the mechanisms of elimination of low-dose hyper-radiosensitivity (HRS) in T-47D cells induced by 0.3 Gy low dose-rate (LDR) priming.

Materials And Methods: The mitotic ratio was measured using mitotic marker histone H3 phosphorylation in LDR primed as well as untreated T-47D cells. The HRS response in unprimed cells receiving medium which was irradiated after being harvested from unprimed cells was measured with or without serum present during cell conditioning. 4,6-benzylidene-D-glucose (BG) was used to inhibit protein synthesis during LDR priming.

Results: LDR primed T-47D cells were HRS-deficient and showed a decrease in mitotic ratio with increasing dose while unprimed, i.e., HRS-competent T-47D cells, showed no decrease in mitotic ratio for doses in the HRS-range. HRS was eliminated in LDR primed cells, in cells receiving medium transfer from LDR primed cells, and in cells receiving LDR irradiated medium harvested from unprimed cells. The efficacy of the transferred medium depended on the presence of serum during cell conditioning. LDR priming eliminated HRS even in the presence of protein synthesis inhibitor BG.

Conclusions: LDR priming of T-47D cells as well as LDR priming of medium conditioned on T-47D cells induce a factor in the medium which cause the early G(2)-checkpoint to be activated in recipient cells by doses normally in the HRS dose-range.
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http://dx.doi.org/10.3109/09553000903242107DOI Listing
December 2009

Gene dosage, expression, and ontology analysis identifies driver genes in the carcinogenesis and chemoradioresistance of cervical cancer.

PLoS Genet 2009 Nov 13;5(11):e1000719. Epub 2009 Nov 13.

Department of Radiation Biology, Norwegian Radium Hospital, Oslo, Norway.

Integrative analysis of gene dosage, expression, and ontology (GO) data was performed to discover driver genes in the carcinogenesis and chemoradioresistance of cervical cancers. Gene dosage and expression profiles of 102 locally advanced cervical cancers were generated by microarray techniques. Fifty-two of these patients were also analyzed with the Illumina expression method to confirm the gene expression results. An independent cohort of 41 patients was used for validation of gene expressions associated with clinical outcome. Statistical analysis identified 29 recurrent gains and losses and 3 losses (on 3p, 13q, 21q) associated with poor outcome after chemoradiotherapy. The intratumor heterogeneity, assessed from the gene dosage profiles, was low for these alterations, showing that they had emerged prior to many other alterations and probably were early events in carcinogenesis. Integration of the alterations with gene expression and GO data identified genes that were regulated by the alterations and revealed five biological processes that were significantly overrepresented among the affected genes: apoptosis, metabolism, macromolecule localization, translation, and transcription. Four genes on 3p (RYBP, GBE1) and 13q (FAM48A, MED4) correlated with outcome at both the gene dosage and expression level and were satisfactorily validated in the independent cohort. These integrated analyses yielded 57 candidate drivers of 24 genetic events, including novel loci responsible for chemoradioresistance. Further mapping of the connections among genetic events, drivers, and biological processes suggested that each individual event stimulates specific processes in carcinogenesis through the coordinated control of multiple genes. The present results may provide novel therapeutic opportunities of both early and advanced stage cervical cancers.
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http://dx.doi.org/10.1371/journal.pgen.1000719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2768783PMC
November 2009

GeneCount: genome-wide calculation of absolute tumor DNA copy numbers from array comparative genomic hybridization data.

Genome Biol 2008 23;9(5):R86. Epub 2008 May 23.

Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Montebello, NO-0310 Oslo, Norway.

Absolute tumor DNA copy numbers can currently be achieved only on a single gene basis by using fluorescence in situ hybridization (FISH). We present GeneCount, a method for genome-wide calculation of absolute copy numbers from clinical array comparative genomic hybridization data. The tumor cell fraction is reliably estimated in the model. Data consistent with FISH results are achieved. We demonstrate significant improvements over existing methods for exploring gene dosages and intratumor copy number heterogeneity in cancers.
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http://dx.doi.org/10.1186/gb-2008-9-5-r86DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2441472PMC
July 2008

Overexpression of human OGG1 in mammalian cells decreases ultraviolet A induced mutagenesis.

Cancer Lett 2008 Aug 11;267(1):18-25. Epub 2008 Apr 11.

Department of Radiation Biology, The Norwegian Radium Hospital, 0310 Oslo, Norway.

7,8-dihydro-8-oxo-guanine (8-oxoG) is a mutagenic DNA lesion that is induced by ultraviolet A (UVA) radiation. 8-oxoG results in increased frequency of GC-->TA transversion mutations. UVA-induced mutant frequency was measured in the guanine phosphoribosyl transferase (gpt) gene of Chinese hamster ovary cells (AS52) that were stably transfected to overexpress the hOGG1 protein, the human DNA repair glycosylase for 8-oxoG. This mutant frequency was compared with ultraviolet A-induced mutant frequency in AS52 cells stably transfected with the same vector without the hOGG1 gene. The mutant frequency was significantly decreased in the hOGG1 overexpressing cells irradiated with 300 and 400 kJ/m2 ultraviolet A radiation, corresponding to 25% and 10% cell survival, respectively. The hOGG1 overexpressing cells repaired oxidative DNA lesions three times faster than the vector only cells as measured by a semi-automated alkaline elution assay using FPG enzyme, the bacterial OGG1 analogue, to cut DNA at oxidative base modifications. Thus, the lower mutation frequency in UVA-induced mutations of the hOGG1 overexpressing cells may be related to the increased repair of 8-oxoG. No GC-->TA mutations were detected in the UVA-irradiated hOGG1 overexpressing cells. The results suggest a link between the 8-oxoG lesion and UVA-induced mutagenesis. We propose that hOGG1 has a role in maintaining genomic stability in mammalian cells after oxidative stress.
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http://dx.doi.org/10.1016/j.canlet.2008.03.002DOI Listing
August 2008

Evaluation of the binding of radiolabeled rituximab to CD20-positive lymphoma cells: an in vitro feasibility study concerning low-dose-rate radioimmunotherapy with the alpha-emitter 227Th.

Cancer Biother Radiopharm 2007 Aug;22(4):469-79

Department of Radiation Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo, Norway.

Radioimmunotherapy (RIT) with the alpha-emitter 227Th is currently under evaluation. 227Th is conjugated to the chimeric anti-CD20 monoclonal antibody rituximab, using the chelator p-isothiocyanato-benzyl-DOTA. In this study, the binding of 227Th-DOTA-p-benzyl-rituximab to three different CD-20-positive lymphoma cell lines, Raji, Rael, and Daudi, were evaluated. Equilibrium and kinetic binding experiments were used to determine binding parameters, including the association and dissociation rate constants, the equilibrium dissociation constants, and the total number of antigens for Raji, Rael, and Daudi cells. There were significant differences between the cell lines with respect to both Kd and the total number of antigens. Rael cells had more than three times as many antigens as the other two cell lines, and the functional Kd found for Rael cells was significantly higher than that found for Raji and Daudi cells. These results were confirmed using flow cytometry. Rituximab was found to be localized in patches on the cell membrane. The findings indicated that 227Th-labeled rituximab has relevant antigen-targeting properties for radioimmunotherapy.
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http://dx.doi.org/10.1089/cbr.2007.371DOI Listing
August 2007
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