Ove Bruland | Haukeland University Hospital, Bergen, Norway (original) (raw)
Papers by Ove Bruland
Investigative Opthalmology & Visual Science, 2015
Congenital stromal corneal dystrophy (CSCD) is an autosomal dominant condition with clouding of t... more Congenital stromal corneal dystrophy (CSCD) is an autosomal dominant condition with clouding of the cornea due to stromal opacities. It is caused by mutations in the decorin gene (DCN) leading to the expression of a truncated form of decorin. In an attempt to replicate this condition in mice, a knock-in mouse strain, 952delT Dcn, was created. Mice were constructed by targeted mutation. Sequencing of genomic DNA confirmed correct genotype. Mouse and human corneas, including corneas from patients with CSCD, and primary keratocyte cultures were subjected to Western blot analysis, transmission electron microscopy, and immunofluorescence microscopy. Histologically, the mice did not show any organ pathology. Corneas were clear, and the electron-lucent deposits observed in CSCD were not present. Furthermore, while nearly equivalent amounts of normal and truncated decorin are present in CSCD corneas, truncated decorin was hardly detectable in the mouse corneas. By immunofluorescence analysis of corneas from 952delT Dcn homozygous mice, decorin was found only in keratocytes. In primary cultures of mouse corneal explants, truncated decorin was retained intracellularly in contrast with human corneal explants where truncated decorin was exported into the culture medium. Immunofluorescence analysis revealed that native mouse decorin localized to the Golgi complex, whereas the truncated decorin accumulated in the endoplasmic reticulum (ER). The ER retention of truncated decorin may explain why the mouse corneas remained clear. The consequences of the decorin mutation are different in mice and humans, and 952delT Dcn knock-in mice are therefore not a suitable model for CSCD.
Objective: Bone tissue engineering (BTE) is evolving as a promising alternative to autologous bon... more Objective: Bone tissue engineering (BTE) is evolving as a promising alternative to autologous bone grafts in the treatment of critical sized bone defects. Bone marrow mesenchymal stem cells (BMSC) in a supportive scaffold , and osteoinductive growth factors can facilitate bone regeneration. The release of osteoinductive factors and the mode of delivery at the defect site are crucial for the biological effects. Recombinant growth factors have short half- life , leading to rapid diffusion and non -availability at the defect site and requires high doses. To combat these limitations, gene transfer employing viral vectors is an emerging new technique for sustained delivery of growth factors . The objective of this study is to examine usefulness of BMSC engineered to express growth factor via adenoviral transfection as a means of growth factor delivery system in BTE. Methods: Adenoviral vector expressing BMP-2 (AdBMP2) was constructed and BMSC were successfully infected and seeded into 3D...
Disease Markers, 2003
Acute intermittent porphyria (AIP), the most common of the acute porphyrias, is caused by mutatio... more Acute intermittent porphyria (AIP), the most common of the acute porphyrias, is caused by mutations in the gene encoding hydroxymethylbilane synthase (HMBS) also called porphobilinogen deaminase (PBGD). The mutation spectrum in the HMBS gene is characterized by a majority of family specific mutations. Among the exceptions are R116W and W198X, with high prevalence in both the Dutch and Swedish populations. These two mutations were also detected in unrelated Norwegian patients. Thus, Norwegian and Swedish patients were haplotyped using closely linked flanking microsatellites and intragenic single nucleotide polymorphisms (SNPs) to see if the high frequency of these two mutations is due to a founder effect. Twelve intragenic SNPs were determined by a method based on fluorescent restriction enzyme fingerprinting single-strand conformation polymorphism (F-REF-SSCP). W198X occurred exclusively on one haplotype in both Norwegian and Swedish patients, showing that it has originated from a common gene source. In contrast, R116W was found on three different haplotypes in three Norwegian families, and in five Swedish families on four or five haplotypes. This extreme haplotype heterogeneity indicates that R116W is a recurrent mutation, maybe explained by the high mutability of CpG dinucleotides. This can also explain why it is the only AIP mutation reported to occur in seven different populations (Norway,
Investigative Opthalmology & Visual Science, 2015
Congenital stromal corneal dystrophy (CSCD) is an autosomal dominant condition with clouding of t... more Congenital stromal corneal dystrophy (CSCD) is an autosomal dominant condition with clouding of the cornea due to stromal opacities. It is caused by mutations in the decorin gene (DCN) leading to the expression of a truncated form of decorin. In an attempt to replicate this condition in mice, a knock-in mouse strain, 952delT Dcn, was created. Mice were constructed by targeted mutation. Sequencing of genomic DNA confirmed correct genotype. Mouse and human corneas, including corneas from patients with CSCD, and primary keratocyte cultures were subjected to Western blot analysis, transmission electron microscopy, and immunofluorescence microscopy. Histologically, the mice did not show any organ pathology. Corneas were clear, and the electron-lucent deposits observed in CSCD were not present. Furthermore, while nearly equivalent amounts of normal and truncated decorin are present in CSCD corneas, truncated decorin was hardly detectable in the mouse corneas. By immunofluorescence analysis of corneas from 952delT Dcn homozygous mice, decorin was found only in keratocytes. In primary cultures of mouse corneal explants, truncated decorin was retained intracellularly in contrast with human corneal explants where truncated decorin was exported into the culture medium. Immunofluorescence analysis revealed that native mouse decorin localized to the Golgi complex, whereas the truncated decorin accumulated in the endoplasmic reticulum (ER). The ER retention of truncated decorin may explain why the mouse corneas remained clear. The consequences of the decorin mutation are different in mice and humans, and 952delT Dcn knock-in mice are therefore not a suitable model for CSCD.
Objective: S100A16 is a recent addition to the S100 protein family. Differential expression of th... more Objective: S100A16 is a recent addition to the S100 protein family. Differential expression of this protein has been reported in many human malignancies. However, its precise biological roles in tumorigenesis are currently unknown. This study aimed to examine the expression pattern and functional role of S100A16 in differentiation of oral squamous cell carcinoma (OSCC). Method: S100A16 mRNA and protein levels were examined in OSCC and normal human oral mucosa (NHOM) respectively by qRT-PCR and immunohistochemistry. Employing retroviral mediated over-expression of S100A16 in oral cancer cell-lines, functional roles of S100A16 was investigated by in vitro and in vivo assays and subsequent molecular analyses. Result: S100A16 expression was found to be down-regulated in OSCCs as compared to NHOMs both at the mRNA and protein levels. Poorly differentiated OSCCs were found to be correlated with low S100A16 mRNA and protein levels. Over-expression of S100A16 was associated with reduced pro...
PLOS ONE, 2015
Myalgic Encephalopathy/Chronic Fatigue Syndrome (ME/CFS) is a disease of unknown etiology. We pre... more Myalgic Encephalopathy/Chronic Fatigue Syndrome (ME/CFS) is a disease of unknown etiology. We previously reported a pilot case series followed by a small, randomized, placebocontrolled phase II study, suggesting that B-cell depletion using the monoclonal anti-CD20 antibody rituximab can yield clinical benefit in ME/CFS.
British journal of cancer, Jan 22, 2011
MicroRNAs (miRNAs) are important regulators of cellular processes and are found to be deregulated... more MicroRNAs (miRNAs) are important regulators of cellular processes and are found to be deregulated in many cancers. We here analysed the miRNA expression in anal carcinomas. In a previous study, we found that our anal carcinoma tumours were divided into two groups based on the expression of E2F-regulated genes. Therefore, we searched for miRNAs that could reproduce this grouping. A global screen of the miRNA population was performed using real-time quantitative PCR (RT-qPCR) array methods and differentially expressed miRNAs were identified. Real-time-qPCR was used to verify the expression levels of selected miRNAs and genes in a larger collection of biopsies. A siRNA-mediated knockdown of human papilloma virus (HPV)16 E7 in a cervical cell line was performed to assess the effect of E7 on miR-15b. The grouping of tumours into two groups based on the expression of E2F-controlled genes was confirmed in a larger collection of anal carcinoma tumours. The expression of miR-15b was shown to...
British journal of cancer, Jan 14, 2012
Our purpose was to investigate if dysregulation of cell adhesion molecules could be linked to pro... more Our purpose was to investigate if dysregulation of cell adhesion molecules could be linked to prognosis in squamous cell carcinomas (SCCs) of the anal region. Protein expression of desmoglein-1 (DSG1), desmocollin-1 (DSC1) and E-cadherin was studied by immunohistochemistry in a cohort of 53 anal carcinoma patients treated by radiation alone or combined with 5-fluorouracil and mitomycin C. Univariate analyses identified, among others, negative membranous DSG1 staining (P=0.009), negative cytoplasmic DSC1 staining (P=0.012) and negative DSG1 (membranous)+negative DSC1 (cytoplasmic) staining (P=0.004) to be associated with improved cancer-specific survival (CSS). On multivariate analyses positive DSG1 (membranous)+DSC1 (cytoplasmic) staining (HR 6.95, P=0.044), large tumour size and lymph node metastases (HR 6.44, P=0.004) and radiation without chemotherapy (HR 6.73 P=0.004) were associated with worse CSS. On univariate analysis, improved disease-free survival was associated with negat...
PloS one, 2011
Chronic fatigue syndrome (CFS) is a disease of unknown aetiology. Major CFS symptom relief during... more Chronic fatigue syndrome (CFS) is a disease of unknown aetiology. Major CFS symptom relief during cancer chemotherapy in a patient with synchronous CFS and lymphoma spurred a pilot study of B-lymphocyte depletion using the anti-CD20 antibody Rituximab, which demonstrated significant clinical response in three CFS patients. In this double-blind, placebo-controlled phase II study (NCT00848692), 30 CFS patients were randomised to either Rituximab 500 mg/m(2) or saline, given twice two weeks apart, with follow-up for 12 months. Xenotropic murine leukemia virus-related virus (XMRV) was not detected in any of the patients. The responses generally affected all CFS symptoms. Major or moderate overall response, defined as lasting improvements in self-reported Fatigue score during follow-up, was seen in 10 out of 15 patients (67%) in the Rituximab group and in two out of 15 patients (13%) in the Placebo group (p = 0.003). Mean response duration within the follow-up period for the 10 responder...
British journal of cancer, Jan 8, 2008
Human papillomavirus (HPV) is a major aetiological agent in anal carcinomas. We here present a st... more Human papillomavirus (HPV) is a major aetiological agent in anal carcinomas. We here present a study of global gene expression using microarray hybridisation in a collection of anal carcinoma biopsies. Quantitative PCR was used to verify expression of selected genes. All biopsies contained integrated DNA of human papillomavirus subtype 16 (HPV16) and expressed HPV16 E7 mRNA. No other subspecies of HPV were detected in these 13 biopsies as assessed by PCR amplification and DNA sequencing. Unsupervised cluster analysis, based on global mRNA expression, divided the tumour biopsies into two distinct groups. Cluster analysis based on a number of high-risk HPV and/or E2F-regulated genes reproduced this biopsy grouping, suggesting that integrated HPV16 substantially influenced global gene expression in approximately half the biopsies studied. The levels of HPV16 E7 mRNA were significantly different between the two groups, but with considerable overlap. Thus, influence on global gene expres...
Molecular and cellular biochemistry, 1998
We have shown that 12-O-tetradecanoylphorbol 13-acetate (TPA) increases protein kinase C (PKC)-me... more We have shown that 12-O-tetradecanoylphorbol 13-acetate (TPA) increases protein kinase C (PKC)-mediated choline transport, incorporation of choline into phosphatidylcholine (PtdCho) and PtdCho degradation by phospholipase D (PLD) in C3H10T1/2 Cl 8 cells. Dual prelabeling experiment using [3H]/[14C]choline indicated that intracellular choline generated from the PLD reaction was not directly recycled to PtdCho synthesis within the cell, and that a large fraction of the choline was transported out of the TPA-treated cells. In contrast, medium derived choline was preferably channeled to PtdCho synthesis. These results indicate that in TPA-treated cells, the choline derived from the PKC-mediated increased PLD activity and the choline newly taken up by the cell behave as two distinctly different metabolic pools.
Neurosurgery, 2010
The vestibular nerve is the predilection site for schwannomas. Few transcriptomic studies have be... more The vestibular nerve is the predilection site for schwannomas. Few transcriptomic studies have been performed on solely sporadic vestibular schwannomas (VSs). To detect genes with altered expression levels in sporadic VSs. We studied 25 VSs and 3 tibial nerves (controls) with the ABI 1700 microarray platform. Significance analysis of microarrays was performed to explore differential gene expression. Selected genes were validated with quantitative reverse transcriptase polymerase chain reaction. A tissue microarray was constructed for immunohistochemistry. Neurofibromatosis type II cDNA was sequenced for mutations. The VSs formed 2 clusters based on the total expression of 23,055 genes. Tumor size, previous Gamma Knife surgery, neurofibromatosis type II mutations, and cystic tumors were distributed equally in both. Significance analysis of microarrays detected 1650 differentially expressed genes. On the top 500 list, several cancer-related genes with an unrecognized role in VSs were down-regulated: CAV1, TGFB3, VCAM1, GLI1, GLI2, PRKAR2B, EPHA4, and FZD1. Immunohistochemistry showed no CAV1 expression in the VSs. The ERK pathway was the central core in the network linking the differentially expressed genes. The previously reported VS candidate genes SPARC, PLAT, and FGF1 were up-regulated. Nineteen of 25 VSs had NF2 mutations. Using microarray technology, we identified novel genes and pathways with a putative role in VSs, confirmed previous candidate genes, and found cancer-related genes with no reported role in VSs. Among these, down-regulation of CAV1 at both the mRNA and protein levels is of particular interest because this tumor suppressor normally is expressed in Schwann cells.
Orphanet Journal of Rare Diseases, 2014
Background: A subset of hereditary cerebellar ataxias is inherited as autosomal recessive traits ... more Background: A subset of hereditary cerebellar ataxias is inherited as autosomal recessive traits (ARCAs). Classification of recessive ataxias due to phenotypic differences in the cerebellum and cerebellar structures is constantly evolving due to new identified disease genes. Recently, reports have linked mutations in genes involved in ubiquitination (RNF216, OTUD4, STUB1) to ARCA with hypogonadism.
Thyroid, 2006
We used cDNA microarrays to study gene expression in fresh frozen papillary thyroid carcinoma (PT... more We used cDNA microarrays to study gene expression in fresh frozen papillary thyroid carcinoma (PTC) specimens. Seven clinically aggressive carcinomas were included, comprising poorly differentiated PTC and tumors with extensive local invasion or synchronous distant metastases. Ten differentiated (classic) papillary thyroid carcinomas (PTC) and non-neoplastic thyroid tissues were also investigated. TaqMan quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry verified the differential gene expression. The B-Raf gene was mutated with a T-->A transversion at nucleotide 1799 (V600E) in 8 of 10 differentiated PTC, and in 4 of 7 aggressive carcinomas. Among genes markedly and equally over-expressed in carcinomas of both the aggressive and classic PtC groups, compared to normal thyroid tissue, were CBP/p300 transactivator (CItED1), fibronectin, growth/differentiation factor 15, potassium inwardly rectifying channel KCNJ2, glutaminyl peptide cyclotransferase, WNT7A, and dipeptidyl peptidase IV. A marked upregulation in carcinomas of P-cadherin mRNA and protein concomitant with E-cadherin downregulation, indicates a possible P-E cadherin "switch" in PTC. The growth factor homologue Nel-like 2, dual specificity phosphatase 5, the serine protease kallikrein 10, and also the tight junction genes claudin 1 and claudin 16, were upregulated in classic PTC but not in aggressive tumors, which may be consistent with altered cell polarity in the dedifferentiated PtC. The aggressive, poorly differentiated PtC group was specifically characterized by marked upregulation of several genes related to cell proliferation such as cell division cycle 2 (CDC2), CDC7, kinesin-like 5, ubiquitin conjugating enzyme E2C, and topoisomerase IIalpha, and by upregulation of genes encoding extracellular matrix proteins such as seprase, extracellular matrix protein 1, and several collagens. These aggressive tumors were also characterized by overexpression of the integrin ligand periostin, and in some biopsies also of osteopontin and of the upstream Rac-regulator dedicator of cytokinesis 10 (DOCK10). These data are interpreted to be consistent with altered cell motility, extracellular matrix remodeling and increased cell proliferation, as important processes in PTC tumor progression.
… et Biophysica Acta (BBA …, 2003
We have studied phospholipase D (PLD) activation in relation to protein kinase C (PKC) and the in... more We have studied phospholipase D (PLD) activation in relation to protein kinase C (PKC) and the involvement of PLD in extracellularly regulated kinase 1 (MAPK) (ERK1) activation and c-fos mRNA expression in C3H/10T1/2 (Cl8) fibroblasts. In these cells, the PLD activity was significantly increased by porcine platelet-derived growth factor (PDGF-BB), phorbol 12-myristate 13-acetate (PMA), and epidermal growth factor (EGF). PLD activation by PDGF-BB and PMA, but not EGF, was inhibited in Cl8 cells expressing the HAbetaC2-1 peptide (Cl8 HAbetaC2-1 cells), with a sequence (betaC2-1) shown to bind receptor for activated C kinase 1 (RACK1) and inhibit c-PKC-mediated cell functions [Science 268 (1995) 247]. A role of alpha-PKC in PLD activation is further underscored by co-immunoprecipitation of alpha-PKC with PLD1 and PLD2 in non-stimulated as well as PMA- and PDGF-BB-stimulated Cl8 cells. However, only PKC in PLD1 precipitates was activated by these agonists, while the PKC in the PLD2 precipitates was constitutively activated. The c-fos mRNA levels in Cl8 cells increased more than 30-fold in response to either PDGF-BB, EGF, or PMA. Approximately 60% inhibition of this increase in c-fos mRNA levels was observed in Cl8 HAbetaC2-1 cells. Formation of phosphatidylbutanol (PtdBut) at the expense of phosphatidic acid (PtdH) in the presence of n-butanol inhibited ERK1 activation and c-fos mRNA expression in PDGF-BB-treated Cl8 cells. ERK activation by PMA was unaffected by n-butanol in Cl8 cells but almost abolished by n-butanol in Cl8 HAbetaC2-1 cells, showing that ERK activation by PMA is heavily dependent on PKC and PLD1. In contrast, ERK activation by EGF in both cell types was not sensitive to n-butanol. These results indicate (1) a role of a functional interaction between the RACK1 scaffolding protein and a alphaPKC-PLD complex for achieving full PLD activity in PDGF-BB- and PMA-stimulated Cl8 cells; (2) PLD-mediated PtdH formation is needed for optimal ERK1 activation by PDGF-BB and maximal increase in c-fos mRNA expression. These findings place PLD as an important component in PDGF-BB- and PMA-stimulated intracellular signalling leading to gene activation in Cl8 cells, while EGF does not require PLD.
Radiotherapy and Oncology, 2008
As low-dose metronomic cyclophosphamide (CTX) and hyperthermia (HT) both exert antitumour effects... more As low-dose metronomic cyclophosphamide (CTX) and hyperthermia (HT) both exert antitumour effects in part through antiangiogenic mechanisms, interactive effects of the two modalities were explored. Subcutaneously implanted rat tumours (BT4An) were treated with CTX 35 mg/kg i.p. three doses a week for two weeks, local water-bath HT yielding mean tumour temperature of 43 degrees C for one hour at day 0, both modalities combined (CTX-HT(0)), or saline. TUNEL assays, immunohistochemical staining of thrombospondin 1 (TSP-1) and real time RT-PCR of TSP-1 mRNA were analysed the first three hours after completed treatment day 0. Metronomic dosed CTX (p=0.006) and HT (p<0.001) both delayed tumour growth. The combined regimen was superior to either modality alone (p<0.001). Complete tumour regressions were observed in CTX (6%), HT (12%), and CTX-HT(0) (41%) treated rats. TSP-1 protein was specifically upregulated in the vascular matrix of tumours receiving CTX (weak), HT (moderate) and CTX-HT(0) (strong). In contrast, reduced expression of TSP-1 protein was observed in tumour cells after HT alone and CTX-HT(0). TUNEL assays indicated induction of apoptosis by HT and CTX-HT(0) 90 minutes after end of the first treatment. A single session of local HT enhances the effects of low-dose metronomic CTX, possibly in part mediated through a differential effect on TSP-1 protein levels in tumour cells and tumour vasculature.
PLoS ONE, 2013
Both S100A14 and S100A16 are members of the multifunctional S100 protein family. Formation of hom... more Both S100A14 and S100A16 are members of the multifunctional S100 protein family. Formation of homo/ heterodimers is considered to be one of the major mechanisms for S100 proteins to execute their diverse cellular functions. By employing a classical Yeast two hybrid (Y-2 H) screen, we identified S100A16 as the single interaction partner of S100A14. This interaction was verified by co-immunoprecipitation, double indirect immunofluorescence and double immunostaining in specimens of oral squamous cell carcinoma and normal oral mucosa. The functional significance of this interaction was examined by employing retroviral mediated over-expression and knock-down of these proteins in several cancer cell-lines. Over-expression and knock-down of S100A14 led to concomitant up-and down-regulation of S100A16 protein in the cell-lines examined. However, there was no up-regulation of S100A16 mRNA upon S100A14 over-expression, indicating that modulation of S100A16 expression was not due to enhanced transcriptional activity but possibly by post-transcriptional regulation. In contrary, over-expression of S100A16 was associated neither with the up-regulation of S100A14 mRNA nor its protein, suggesting a unidirectional regulation between S100A14 and S100A16. Cellular treatment with protein synthesis inhibitor cycloheximide demonstrated a time-dependent intracellular degradation of both S100A16 and S100A14 proteins. Additionally, regulation of S100A16 and S100A14 degradation was found to be independent of the classical proteasomal and lysosomal pathways of protein degradation. Further studies will therefore be necessary to understand the functional significance of this interaction and the mechanisms on how S100A14 is involved in the regulation of S100A16 expression.
Oral Oncology, 2012
Altered expression of S100A14 has been reported in various human cancers including oral squamous ... more Altered expression of S100A14 has been reported in various human cancers including oral squamous cell carcinomas (OSCCs). Its biological functions in carcinogenesis, however, are largely unknown. This study aimed to investigate the functional role of S100A14 in tumor cell proliferation and its possible functional association with p53. S100A14 protein was found to be gradually down-regulated during the transition from normal to dysplastic and carcinoma cells in an in vitro human OSCC progression model. When over-expressed by employing retroviral expression vector, S100A14 inhibited proliferation of CaLH3 and OSCC1, OSCC cell-lines harboring wild type (wt) p53, by inducing G1-arrest. This G1-arrest correlated with up-regulation of p21 both in the CaLH3 and OSCC1 cell-lines. shRNA mediated silencing of p53 led to partial suppression of p21 in S100A14 over-expressing CaLH3 cells, indicating that p21 up-regulation was, at least, partly dependent on p53. We further demonstrated that nuclear accumulation of p53 occurred with over-expression of S100A14 in CaLH3 cells. Our data suggest a novel role of S100A14 in OSCC cell proliferation by inducing G1-arrest and also indicate a functional link between S100A14 and the tumor suppressor protein p53.
Investigative Opthalmology & Visual Science, 2015
Congenital stromal corneal dystrophy (CSCD) is an autosomal dominant condition with clouding of t... more Congenital stromal corneal dystrophy (CSCD) is an autosomal dominant condition with clouding of the cornea due to stromal opacities. It is caused by mutations in the decorin gene (DCN) leading to the expression of a truncated form of decorin. In an attempt to replicate this condition in mice, a knock-in mouse strain, 952delT Dcn, was created. Mice were constructed by targeted mutation. Sequencing of genomic DNA confirmed correct genotype. Mouse and human corneas, including corneas from patients with CSCD, and primary keratocyte cultures were subjected to Western blot analysis, transmission electron microscopy, and immunofluorescence microscopy. Histologically, the mice did not show any organ pathology. Corneas were clear, and the electron-lucent deposits observed in CSCD were not present. Furthermore, while nearly equivalent amounts of normal and truncated decorin are present in CSCD corneas, truncated decorin was hardly detectable in the mouse corneas. By immunofluorescence analysis of corneas from 952delT Dcn homozygous mice, decorin was found only in keratocytes. In primary cultures of mouse corneal explants, truncated decorin was retained intracellularly in contrast with human corneal explants where truncated decorin was exported into the culture medium. Immunofluorescence analysis revealed that native mouse decorin localized to the Golgi complex, whereas the truncated decorin accumulated in the endoplasmic reticulum (ER). The ER retention of truncated decorin may explain why the mouse corneas remained clear. The consequences of the decorin mutation are different in mice and humans, and 952delT Dcn knock-in mice are therefore not a suitable model for CSCD.
Objective: Bone tissue engineering (BTE) is evolving as a promising alternative to autologous bon... more Objective: Bone tissue engineering (BTE) is evolving as a promising alternative to autologous bone grafts in the treatment of critical sized bone defects. Bone marrow mesenchymal stem cells (BMSC) in a supportive scaffold , and osteoinductive growth factors can facilitate bone regeneration. The release of osteoinductive factors and the mode of delivery at the defect site are crucial for the biological effects. Recombinant growth factors have short half- life , leading to rapid diffusion and non -availability at the defect site and requires high doses. To combat these limitations, gene transfer employing viral vectors is an emerging new technique for sustained delivery of growth factors . The objective of this study is to examine usefulness of BMSC engineered to express growth factor via adenoviral transfection as a means of growth factor delivery system in BTE. Methods: Adenoviral vector expressing BMP-2 (AdBMP2) was constructed and BMSC were successfully infected and seeded into 3D...
Disease Markers, 2003
Acute intermittent porphyria (AIP), the most common of the acute porphyrias, is caused by mutatio... more Acute intermittent porphyria (AIP), the most common of the acute porphyrias, is caused by mutations in the gene encoding hydroxymethylbilane synthase (HMBS) also called porphobilinogen deaminase (PBGD). The mutation spectrum in the HMBS gene is characterized by a majority of family specific mutations. Among the exceptions are R116W and W198X, with high prevalence in both the Dutch and Swedish populations. These two mutations were also detected in unrelated Norwegian patients. Thus, Norwegian and Swedish patients were haplotyped using closely linked flanking microsatellites and intragenic single nucleotide polymorphisms (SNPs) to see if the high frequency of these two mutations is due to a founder effect. Twelve intragenic SNPs were determined by a method based on fluorescent restriction enzyme fingerprinting single-strand conformation polymorphism (F-REF-SSCP). W198X occurred exclusively on one haplotype in both Norwegian and Swedish patients, showing that it has originated from a common gene source. In contrast, R116W was found on three different haplotypes in three Norwegian families, and in five Swedish families on four or five haplotypes. This extreme haplotype heterogeneity indicates that R116W is a recurrent mutation, maybe explained by the high mutability of CpG dinucleotides. This can also explain why it is the only AIP mutation reported to occur in seven different populations (Norway,
Investigative Opthalmology & Visual Science, 2015
Congenital stromal corneal dystrophy (CSCD) is an autosomal dominant condition with clouding of t... more Congenital stromal corneal dystrophy (CSCD) is an autosomal dominant condition with clouding of the cornea due to stromal opacities. It is caused by mutations in the decorin gene (DCN) leading to the expression of a truncated form of decorin. In an attempt to replicate this condition in mice, a knock-in mouse strain, 952delT Dcn, was created. Mice were constructed by targeted mutation. Sequencing of genomic DNA confirmed correct genotype. Mouse and human corneas, including corneas from patients with CSCD, and primary keratocyte cultures were subjected to Western blot analysis, transmission electron microscopy, and immunofluorescence microscopy. Histologically, the mice did not show any organ pathology. Corneas were clear, and the electron-lucent deposits observed in CSCD were not present. Furthermore, while nearly equivalent amounts of normal and truncated decorin are present in CSCD corneas, truncated decorin was hardly detectable in the mouse corneas. By immunofluorescence analysis of corneas from 952delT Dcn homozygous mice, decorin was found only in keratocytes. In primary cultures of mouse corneal explants, truncated decorin was retained intracellularly in contrast with human corneal explants where truncated decorin was exported into the culture medium. Immunofluorescence analysis revealed that native mouse decorin localized to the Golgi complex, whereas the truncated decorin accumulated in the endoplasmic reticulum (ER). The ER retention of truncated decorin may explain why the mouse corneas remained clear. The consequences of the decorin mutation are different in mice and humans, and 952delT Dcn knock-in mice are therefore not a suitable model for CSCD.
Objective: S100A16 is a recent addition to the S100 protein family. Differential expression of th... more Objective: S100A16 is a recent addition to the S100 protein family. Differential expression of this protein has been reported in many human malignancies. However, its precise biological roles in tumorigenesis are currently unknown. This study aimed to examine the expression pattern and functional role of S100A16 in differentiation of oral squamous cell carcinoma (OSCC). Method: S100A16 mRNA and protein levels were examined in OSCC and normal human oral mucosa (NHOM) respectively by qRT-PCR and immunohistochemistry. Employing retroviral mediated over-expression of S100A16 in oral cancer cell-lines, functional roles of S100A16 was investigated by in vitro and in vivo assays and subsequent molecular analyses. Result: S100A16 expression was found to be down-regulated in OSCCs as compared to NHOMs both at the mRNA and protein levels. Poorly differentiated OSCCs were found to be correlated with low S100A16 mRNA and protein levels. Over-expression of S100A16 was associated with reduced pro...
PLOS ONE, 2015
Myalgic Encephalopathy/Chronic Fatigue Syndrome (ME/CFS) is a disease of unknown etiology. We pre... more Myalgic Encephalopathy/Chronic Fatigue Syndrome (ME/CFS) is a disease of unknown etiology. We previously reported a pilot case series followed by a small, randomized, placebocontrolled phase II study, suggesting that B-cell depletion using the monoclonal anti-CD20 antibody rituximab can yield clinical benefit in ME/CFS.
British journal of cancer, Jan 22, 2011
MicroRNAs (miRNAs) are important regulators of cellular processes and are found to be deregulated... more MicroRNAs (miRNAs) are important regulators of cellular processes and are found to be deregulated in many cancers. We here analysed the miRNA expression in anal carcinomas. In a previous study, we found that our anal carcinoma tumours were divided into two groups based on the expression of E2F-regulated genes. Therefore, we searched for miRNAs that could reproduce this grouping. A global screen of the miRNA population was performed using real-time quantitative PCR (RT-qPCR) array methods and differentially expressed miRNAs were identified. Real-time-qPCR was used to verify the expression levels of selected miRNAs and genes in a larger collection of biopsies. A siRNA-mediated knockdown of human papilloma virus (HPV)16 E7 in a cervical cell line was performed to assess the effect of E7 on miR-15b. The grouping of tumours into two groups based on the expression of E2F-controlled genes was confirmed in a larger collection of anal carcinoma tumours. The expression of miR-15b was shown to...
British journal of cancer, Jan 14, 2012
Our purpose was to investigate if dysregulation of cell adhesion molecules could be linked to pro... more Our purpose was to investigate if dysregulation of cell adhesion molecules could be linked to prognosis in squamous cell carcinomas (SCCs) of the anal region. Protein expression of desmoglein-1 (DSG1), desmocollin-1 (DSC1) and E-cadherin was studied by immunohistochemistry in a cohort of 53 anal carcinoma patients treated by radiation alone or combined with 5-fluorouracil and mitomycin C. Univariate analyses identified, among others, negative membranous DSG1 staining (P=0.009), negative cytoplasmic DSC1 staining (P=0.012) and negative DSG1 (membranous)+negative DSC1 (cytoplasmic) staining (P=0.004) to be associated with improved cancer-specific survival (CSS). On multivariate analyses positive DSG1 (membranous)+DSC1 (cytoplasmic) staining (HR 6.95, P=0.044), large tumour size and lymph node metastases (HR 6.44, P=0.004) and radiation without chemotherapy (HR 6.73 P=0.004) were associated with worse CSS. On univariate analysis, improved disease-free survival was associated with negat...
PloS one, 2011
Chronic fatigue syndrome (CFS) is a disease of unknown aetiology. Major CFS symptom relief during... more Chronic fatigue syndrome (CFS) is a disease of unknown aetiology. Major CFS symptom relief during cancer chemotherapy in a patient with synchronous CFS and lymphoma spurred a pilot study of B-lymphocyte depletion using the anti-CD20 antibody Rituximab, which demonstrated significant clinical response in three CFS patients. In this double-blind, placebo-controlled phase II study (NCT00848692), 30 CFS patients were randomised to either Rituximab 500 mg/m(2) or saline, given twice two weeks apart, with follow-up for 12 months. Xenotropic murine leukemia virus-related virus (XMRV) was not detected in any of the patients. The responses generally affected all CFS symptoms. Major or moderate overall response, defined as lasting improvements in self-reported Fatigue score during follow-up, was seen in 10 out of 15 patients (67%) in the Rituximab group and in two out of 15 patients (13%) in the Placebo group (p = 0.003). Mean response duration within the follow-up period for the 10 responder...
British journal of cancer, Jan 8, 2008
Human papillomavirus (HPV) is a major aetiological agent in anal carcinomas. We here present a st... more Human papillomavirus (HPV) is a major aetiological agent in anal carcinomas. We here present a study of global gene expression using microarray hybridisation in a collection of anal carcinoma biopsies. Quantitative PCR was used to verify expression of selected genes. All biopsies contained integrated DNA of human papillomavirus subtype 16 (HPV16) and expressed HPV16 E7 mRNA. No other subspecies of HPV were detected in these 13 biopsies as assessed by PCR amplification and DNA sequencing. Unsupervised cluster analysis, based on global mRNA expression, divided the tumour biopsies into two distinct groups. Cluster analysis based on a number of high-risk HPV and/or E2F-regulated genes reproduced this biopsy grouping, suggesting that integrated HPV16 substantially influenced global gene expression in approximately half the biopsies studied. The levels of HPV16 E7 mRNA were significantly different between the two groups, but with considerable overlap. Thus, influence on global gene expres...
Molecular and cellular biochemistry, 1998
We have shown that 12-O-tetradecanoylphorbol 13-acetate (TPA) increases protein kinase C (PKC)-me... more We have shown that 12-O-tetradecanoylphorbol 13-acetate (TPA) increases protein kinase C (PKC)-mediated choline transport, incorporation of choline into phosphatidylcholine (PtdCho) and PtdCho degradation by phospholipase D (PLD) in C3H10T1/2 Cl 8 cells. Dual prelabeling experiment using [3H]/[14C]choline indicated that intracellular choline generated from the PLD reaction was not directly recycled to PtdCho synthesis within the cell, and that a large fraction of the choline was transported out of the TPA-treated cells. In contrast, medium derived choline was preferably channeled to PtdCho synthesis. These results indicate that in TPA-treated cells, the choline derived from the PKC-mediated increased PLD activity and the choline newly taken up by the cell behave as two distinctly different metabolic pools.
Neurosurgery, 2010
The vestibular nerve is the predilection site for schwannomas. Few transcriptomic studies have be... more The vestibular nerve is the predilection site for schwannomas. Few transcriptomic studies have been performed on solely sporadic vestibular schwannomas (VSs). To detect genes with altered expression levels in sporadic VSs. We studied 25 VSs and 3 tibial nerves (controls) with the ABI 1700 microarray platform. Significance analysis of microarrays was performed to explore differential gene expression. Selected genes were validated with quantitative reverse transcriptase polymerase chain reaction. A tissue microarray was constructed for immunohistochemistry. Neurofibromatosis type II cDNA was sequenced for mutations. The VSs formed 2 clusters based on the total expression of 23,055 genes. Tumor size, previous Gamma Knife surgery, neurofibromatosis type II mutations, and cystic tumors were distributed equally in both. Significance analysis of microarrays detected 1650 differentially expressed genes. On the top 500 list, several cancer-related genes with an unrecognized role in VSs were down-regulated: CAV1, TGFB3, VCAM1, GLI1, GLI2, PRKAR2B, EPHA4, and FZD1. Immunohistochemistry showed no CAV1 expression in the VSs. The ERK pathway was the central core in the network linking the differentially expressed genes. The previously reported VS candidate genes SPARC, PLAT, and FGF1 were up-regulated. Nineteen of 25 VSs had NF2 mutations. Using microarray technology, we identified novel genes and pathways with a putative role in VSs, confirmed previous candidate genes, and found cancer-related genes with no reported role in VSs. Among these, down-regulation of CAV1 at both the mRNA and protein levels is of particular interest because this tumor suppressor normally is expressed in Schwann cells.
Orphanet Journal of Rare Diseases, 2014
Background: A subset of hereditary cerebellar ataxias is inherited as autosomal recessive traits ... more Background: A subset of hereditary cerebellar ataxias is inherited as autosomal recessive traits (ARCAs). Classification of recessive ataxias due to phenotypic differences in the cerebellum and cerebellar structures is constantly evolving due to new identified disease genes. Recently, reports have linked mutations in genes involved in ubiquitination (RNF216, OTUD4, STUB1) to ARCA with hypogonadism.
Thyroid, 2006
We used cDNA microarrays to study gene expression in fresh frozen papillary thyroid carcinoma (PT... more We used cDNA microarrays to study gene expression in fresh frozen papillary thyroid carcinoma (PTC) specimens. Seven clinically aggressive carcinomas were included, comprising poorly differentiated PTC and tumors with extensive local invasion or synchronous distant metastases. Ten differentiated (classic) papillary thyroid carcinomas (PTC) and non-neoplastic thyroid tissues were also investigated. TaqMan quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry verified the differential gene expression. The B-Raf gene was mutated with a T-->A transversion at nucleotide 1799 (V600E) in 8 of 10 differentiated PTC, and in 4 of 7 aggressive carcinomas. Among genes markedly and equally over-expressed in carcinomas of both the aggressive and classic PtC groups, compared to normal thyroid tissue, were CBP/p300 transactivator (CItED1), fibronectin, growth/differentiation factor 15, potassium inwardly rectifying channel KCNJ2, glutaminyl peptide cyclotransferase, WNT7A, and dipeptidyl peptidase IV. A marked upregulation in carcinomas of P-cadherin mRNA and protein concomitant with E-cadherin downregulation, indicates a possible P-E cadherin "switch" in PTC. The growth factor homologue Nel-like 2, dual specificity phosphatase 5, the serine protease kallikrein 10, and also the tight junction genes claudin 1 and claudin 16, were upregulated in classic PTC but not in aggressive tumors, which may be consistent with altered cell polarity in the dedifferentiated PtC. The aggressive, poorly differentiated PtC group was specifically characterized by marked upregulation of several genes related to cell proliferation such as cell division cycle 2 (CDC2), CDC7, kinesin-like 5, ubiquitin conjugating enzyme E2C, and topoisomerase IIalpha, and by upregulation of genes encoding extracellular matrix proteins such as seprase, extracellular matrix protein 1, and several collagens. These aggressive tumors were also characterized by overexpression of the integrin ligand periostin, and in some biopsies also of osteopontin and of the upstream Rac-regulator dedicator of cytokinesis 10 (DOCK10). These data are interpreted to be consistent with altered cell motility, extracellular matrix remodeling and increased cell proliferation, as important processes in PTC tumor progression.
… et Biophysica Acta (BBA …, 2003
We have studied phospholipase D (PLD) activation in relation to protein kinase C (PKC) and the in... more We have studied phospholipase D (PLD) activation in relation to protein kinase C (PKC) and the involvement of PLD in extracellularly regulated kinase 1 (MAPK) (ERK1) activation and c-fos mRNA expression in C3H/10T1/2 (Cl8) fibroblasts. In these cells, the PLD activity was significantly increased by porcine platelet-derived growth factor (PDGF-BB), phorbol 12-myristate 13-acetate (PMA), and epidermal growth factor (EGF). PLD activation by PDGF-BB and PMA, but not EGF, was inhibited in Cl8 cells expressing the HAbetaC2-1 peptide (Cl8 HAbetaC2-1 cells), with a sequence (betaC2-1) shown to bind receptor for activated C kinase 1 (RACK1) and inhibit c-PKC-mediated cell functions [Science 268 (1995) 247]. A role of alpha-PKC in PLD activation is further underscored by co-immunoprecipitation of alpha-PKC with PLD1 and PLD2 in non-stimulated as well as PMA- and PDGF-BB-stimulated Cl8 cells. However, only PKC in PLD1 precipitates was activated by these agonists, while the PKC in the PLD2 precipitates was constitutively activated. The c-fos mRNA levels in Cl8 cells increased more than 30-fold in response to either PDGF-BB, EGF, or PMA. Approximately 60% inhibition of this increase in c-fos mRNA levels was observed in Cl8 HAbetaC2-1 cells. Formation of phosphatidylbutanol (PtdBut) at the expense of phosphatidic acid (PtdH) in the presence of n-butanol inhibited ERK1 activation and c-fos mRNA expression in PDGF-BB-treated Cl8 cells. ERK activation by PMA was unaffected by n-butanol in Cl8 cells but almost abolished by n-butanol in Cl8 HAbetaC2-1 cells, showing that ERK activation by PMA is heavily dependent on PKC and PLD1. In contrast, ERK activation by EGF in both cell types was not sensitive to n-butanol. These results indicate (1) a role of a functional interaction between the RACK1 scaffolding protein and a alphaPKC-PLD complex for achieving full PLD activity in PDGF-BB- and PMA-stimulated Cl8 cells; (2) PLD-mediated PtdH formation is needed for optimal ERK1 activation by PDGF-BB and maximal increase in c-fos mRNA expression. These findings place PLD as an important component in PDGF-BB- and PMA-stimulated intracellular signalling leading to gene activation in Cl8 cells, while EGF does not require PLD.
Radiotherapy and Oncology, 2008
As low-dose metronomic cyclophosphamide (CTX) and hyperthermia (HT) both exert antitumour effects... more As low-dose metronomic cyclophosphamide (CTX) and hyperthermia (HT) both exert antitumour effects in part through antiangiogenic mechanisms, interactive effects of the two modalities were explored. Subcutaneously implanted rat tumours (BT4An) were treated with CTX 35 mg/kg i.p. three doses a week for two weeks, local water-bath HT yielding mean tumour temperature of 43 degrees C for one hour at day 0, both modalities combined (CTX-HT(0)), or saline. TUNEL assays, immunohistochemical staining of thrombospondin 1 (TSP-1) and real time RT-PCR of TSP-1 mRNA were analysed the first three hours after completed treatment day 0. Metronomic dosed CTX (p=0.006) and HT (p<0.001) both delayed tumour growth. The combined regimen was superior to either modality alone (p<0.001). Complete tumour regressions were observed in CTX (6%), HT (12%), and CTX-HT(0) (41%) treated rats. TSP-1 protein was specifically upregulated in the vascular matrix of tumours receiving CTX (weak), HT (moderate) and CTX-HT(0) (strong). In contrast, reduced expression of TSP-1 protein was observed in tumour cells after HT alone and CTX-HT(0). TUNEL assays indicated induction of apoptosis by HT and CTX-HT(0) 90 minutes after end of the first treatment. A single session of local HT enhances the effects of low-dose metronomic CTX, possibly in part mediated through a differential effect on TSP-1 protein levels in tumour cells and tumour vasculature.
PLoS ONE, 2013
Both S100A14 and S100A16 are members of the multifunctional S100 protein family. Formation of hom... more Both S100A14 and S100A16 are members of the multifunctional S100 protein family. Formation of homo/ heterodimers is considered to be one of the major mechanisms for S100 proteins to execute their diverse cellular functions. By employing a classical Yeast two hybrid (Y-2 H) screen, we identified S100A16 as the single interaction partner of S100A14. This interaction was verified by co-immunoprecipitation, double indirect immunofluorescence and double immunostaining in specimens of oral squamous cell carcinoma and normal oral mucosa. The functional significance of this interaction was examined by employing retroviral mediated over-expression and knock-down of these proteins in several cancer cell-lines. Over-expression and knock-down of S100A14 led to concomitant up-and down-regulation of S100A16 protein in the cell-lines examined. However, there was no up-regulation of S100A16 mRNA upon S100A14 over-expression, indicating that modulation of S100A16 expression was not due to enhanced transcriptional activity but possibly by post-transcriptional regulation. In contrary, over-expression of S100A16 was associated neither with the up-regulation of S100A14 mRNA nor its protein, suggesting a unidirectional regulation between S100A14 and S100A16. Cellular treatment with protein synthesis inhibitor cycloheximide demonstrated a time-dependent intracellular degradation of both S100A16 and S100A14 proteins. Additionally, regulation of S100A16 and S100A14 degradation was found to be independent of the classical proteasomal and lysosomal pathways of protein degradation. Further studies will therefore be necessary to understand the functional significance of this interaction and the mechanisms on how S100A14 is involved in the regulation of S100A16 expression.
Oral Oncology, 2012
Altered expression of S100A14 has been reported in various human cancers including oral squamous ... more Altered expression of S100A14 has been reported in various human cancers including oral squamous cell carcinomas (OSCCs). Its biological functions in carcinogenesis, however, are largely unknown. This study aimed to investigate the functional role of S100A14 in tumor cell proliferation and its possible functional association with p53. S100A14 protein was found to be gradually down-regulated during the transition from normal to dysplastic and carcinoma cells in an in vitro human OSCC progression model. When over-expressed by employing retroviral expression vector, S100A14 inhibited proliferation of CaLH3 and OSCC1, OSCC cell-lines harboring wild type (wt) p53, by inducing G1-arrest. This G1-arrest correlated with up-regulation of p21 both in the CaLH3 and OSCC1 cell-lines. shRNA mediated silencing of p53 led to partial suppression of p21 in S100A14 over-expressing CaLH3 cells, indicating that p21 up-regulation was, at least, partly dependent on p53. We further demonstrated that nuclear accumulation of p53 occurred with over-expression of S100A14 in CaLH3 cells. Our data suggest a novel role of S100A14 in OSCC cell proliferation by inducing G1-arrest and also indicate a functional link between S100A14 and the tumor suppressor protein p53.