Trond Flægstad

Tumour-suppressor microRNAs let-7 and mir-101 target the proto-oncogene MYCN and inhibit cell proliferation in MYCN-amplified neuroblastoma

Background:MicroRNAs (miRNAs) regulate expression of many cancer-related genes through posttranscriptional repression of their mRNAs. In this study we investigate the proto-oncogene MYCN as a target for miRNA regulation.Methods:A luciferase reporter assay was used to investigate software-predicted miRNA target sites in the 3′-untranslated region (3′UTR) of MYCN. The miRNAs were overexpressed in cell lines by transfection of miRNA mimics or miRNA-expressing plasmids. Mutation of the target sites was used to validate MYCN 3′UTR as a direct target of several miRNAs. To measure miRNA-mediated suppression of endogenous N-myc protein, inhibition of proliferation and inhibition of clonogenic growth, miRNAs were overexpressed in a MYCN-amplified neuroblastoma cell line.Results:The results from this study show that MYCN is targeted by several miRNAs. In addition to the previously shown mir-34a/c, we experimentally validate mir-449, mir-19a/b, mir-29a/b/c, mir-101 and let-7e/mir-202 as direct MYCN-targeting miRNAs. These miRNAs were able to suppress endogenous N-myc protein in a MYCN-amplified neuroblastoma cell line. The let-7e and mir-202 were strong negative regulators of MYCN expression. The mir-101 and the let-7 family miRNAs let-7e and mir-202 inhibited proliferation and clonogenic growth when overexpressed in Kelly cells.Conclusion:The tumour-suppressor miRNAs let-7 and mir-101 target MYCN and inhibit proliferation and clonogenic growth of MYCN-amplified neuroblastoma cells.

Biofilm Formation by Staphylococcus haemolyticus

ABSTRACT Infections due to coagulase-negative staphylococci (CoNS) most frequently occur after the implantation of medical devices and are attributed to the biofilm-forming potential of CoNS. Staphylococcus haemolyticus is the second most frequently isolated CoNS from patients with hospital-acquired infections. There is only limited knowledge of the nature of S. haemolyticus biofilms. The aim of this study was to characterize S. haemolyticus biofilm formation. We analyzed the biofilm-forming capacities of 72 clinical S. haemolyticus isolates. A detachment assay with NaIO4, proteinase K, or DNase was used to determine the main biofilm components. Biofilm-associated genes, including the ica operon, were analyzed by PCR, and the gene products were sequenced. Confocal laser scanning microscopy (CLSM) was used to elucidate the biofilm structure. Fifty-three isolates (74%) produced biofilms after growth in Trypticase soy broth (TSB) with glucose, but only 22 (31%) produced biofilms after growth in TSB with NaCl. It was necessary to dissolve the biofilm in ethanol-acetone to measure the optical density of the full biofilm mass. DNase, proteinase K, and NaIO4 caused biofilm detachment for 100%, 98%, and 38% of the isolates, respectively. icaRADBC and polysaccharide intercellular adhesin (PIA) production were found in only two isolates. CLSM indicated that the biofilm structure of S. haemolyticus clearly differs from that of S. epidermidis. We conclude that biofilm formation is a common phenotype in clinical S. haemolyticus isolates. In contrast to S. epidermidis, proteins and extracellular DNA are of functional relevance for biofilm accumulation, whereas PIA plays only a minor role. The induction of biofilm formation and determination of the biofilm mass also needed to be optimized for S. haemolyticus.

The antimicrobial peptide, lactoferricin B, is cytotoxic to neuroblastoma cells in vitro and inhibits xenograft growth in vivo.

Antimicrobial peptides have been shown to exert cytotoxic activity towards cancer cells through their ability to interact with negatively charged cell membranes. In this study the cytotoxic effect of the antimicrobial peptide, LfcinB was tested in a panel of human neuroblastoma cell lines. LfcinB displayed a selective cytotoxic activity against both MYCN‐amplified and non‐MYCN‐amplified cell lines. Non‐transformed fibroblasts were not substantially affected by LfcinB. Treatment of neuroblastoma cells with LfcinB induced rapid destabilization of the cytoplasmic membrane and formation of membrane blebs. Depolarization of the mitochondria membranes and irreversible changes in the mitochondria morphology was also evident. Immuno‐ and fluorescence‐labeled LfcinB revealed that the peptide co‐localized with mitochondria. Furthermore, treatment of neuroblastoma cells with LfcinB induced cleavage of caspase‐6, ‐7 and ‐9 followed by cell death. However, neither addition of the pan‐caspase inhibitor, zVAD‐fmk, or specific caspase inhibitors could reverse the cytotoxic effect induced by LfcinB. Treatment of established SH‐SY‐5Y neuroblastoma xenografts with repeated injections of LfcinB resulted in significant tumor growth inhibition. These results revealed a selective destabilizing effect of LfcinB on two important targets in the neuroblastoma cells, the cytoplasmic‐ and the mitochondria membrane.

Genome-wide signatures of differential DNA methylation in pediatric acute lymphoblastic leukemia

BackgroundAlthough aberrant DNA methylation has been observed previously in acute lymphoblastic leukemia (ALL), the patterns of differential methylation have not been comprehensively determined in all subtypes of ALL on a genome-wide scale. The relationship between DNA methylation, cytogenetic background, drug resistance and relapse in ALL is poorly understood.ResultsWe surveyed the DNA methylation levels of 435,941 CpG sites in samples from 764 children at diagnosis of ALL and from 27 children at relapse. This survey uncovered four characteristic methylation signatures. First, compared with control blood cells, the methylomes of ALL cells shared 9,406 predominantly hypermethylated CpG sites, independent of cytogenetic background. Second, each cytogenetic subtype of ALL displayed a unique set of hyper- and hypomethylated CpG sites. The CpG sites that constituted these two signatures differed in their functional genomic enrichment to regions with marks of active or repressed chromatin. Third, we identified subtype-specific differential methylation in promoter and enhancer regions that were strongly correlated with gene expression. Fourth, a set of 6,612 CpG sites was predominantly hypermethylated in ALL cells at relapse, compared with matched samples at diagnosis. Analysis of relapse-free survival identified CpG sites with subtype-specific differential methylation that divided the patients into different risk groups, depending on their methylation status.ConclusionsOur results suggest an important biological role for DNA methylation in the differences between ALL subtypes and in their clinical outcome after treatment.

DNA methylation for subtype classification and prediction of treatment outcome in patients with childhood acute lymphoblastic leukemia

Despite improvements in the prognosis of childhood acute lymphoblastic leukemia (ALL), subgroups of patients would benefit from alternative treatment approaches. Our aim was to identify genes with DNA methylation profiles that could identify such groups. We determined the methylation levels of 1320 CpG sites in regulatory regions of 416 genes in cells from 401 children diagnosed with ALL. Hierarchical clustering of 300 CpG sites distinguished between T-lineage ALL and B-cell precursor (BCP) ALL and between the main cytogenetic subtypes of BCP ALL. It also stratified patients with high hyperdiploidy and t(12;21) ALL into 2 subgroups with different probability of relapse. By using supervised learning, we constructed multivariate classifiers by external cross-validation procedures. We identified 40 genes that consistently contributed to accurate discrimination between the main subtypes of BCP ALL and gene sets that discriminated between subtypes of ALL and between ALL and controls in pairwise classification analyses. We also identified 20 individual genes with DNA methylation levels that predicted relapse of leukemia. Thus, methylation analysis should be explored as a method to improve stratification of ALL patients. The genes highlighted in our study are not enriched to specific pathways, but the gene expression levels are inversely correlated to the methylation levels.

Coagulase-negative staphylococcal sepsis in neonates. Association between antibiotic resistance, biofilm formation and the host inflammatory response.

Background: Coagulase-negative staphylococci (CoNS) are the most prevalent pathogens causing late onset sepsis in neonates. They are often multiresistant to antibiotics, and the ability to form biofilm is considered their main virulence determinant. Methods: During a 12-year period, we identified 150 neonates having 164 suspected septic episodes with growth of CoNS in blood culture. We examined the relationship between antibiotic resistance, phenotypic biofilm production and genetic determinants for biofilm formation in different CoNS species and their correlation with neonatal inflammatory response. Results: Eighty-five episodes were classified as true sepsis, and 79 episodes of CoNS growth in blood culture were considered contaminations. Sixty-one percent of Staphylococcus epidermidis isolates produced biofilm compared with 26% of CoNS non-epidermidis (P < 0.001). We observed no difference in phenotypic biofilm production or genetic determinants for biofilm formation between invasive isolates and contaminants. C-reactive protein levels as a marker of inflammatory response were higher in CoNS sepsis caused by methicillin and aminoglycoside resistant versus susceptible isolates (P = 0.031). In contrast, there was a significant association between a lower C-reactive protein response and biofilm-positive isolates (P = 0.018). Antibiotic resistance was significantly correlated with biofilm production in S. epidermidis, but not in other CoNS species. Conclusions: CoNS sepsis with biofilm-forming strains was associated with a decreased host inflammatory response, potentially limiting the immune system to counteract the infection. The impact of antibiotic resistance and virulence determinants on clinical outcome of neonatal CoNS sepsis warrants additional clinical studies.

Allele-specific gene expression patterns in primary leukemic cells reveal regulation of gene expression by CpG site methylation

To identify genes that are regulated by cis-acting functional elements in acute lymphoblastic leukemia (ALL) we determined the allele-specific expression (ASE) levels of 2, 529 genes by genotyping a genome-wide panel of single nucleotide polymorphisms in RNA and DNA from bone marrow and blood samples of 197 children with ALL. Using a reproducible, quantitative genotyping method and stringent criteria for scoring ASE, we found that 16% of the analyzed genes display ASE in multiple ALL cell samples. For most of the genes, the level of ASE varied largely between the samples, from 1.4-fold overexpression of one allele to apparent monoallelic expression. For genes exhibiting ASE, 55% displayed bidirectional ASE in which overexpression of either of the two SNP alleles occurred. For bidirectional ASE we also observed overall higher levels of ASE and correlation with the methylation level of these sites. Our results demonstrate that CpG site methylation is one of the factors that regulates gene expression in ALL cells.

High in vitro antimicrobial activity of synthetic antimicrobial peptidomimetics against staphylococcal biofilms

OBJECTIVES The aim of the study was to investigate the antimicrobial effect of different antibiotics and synthetic antimicrobial peptidomimetics (SAMPs) on staphylococcal biofilms. METHODS Biofilms of six staphylococcal strains (two Staphylococcus haemolyticus, two Staphylococcus epidermidis and two Staphylococcus aureus isolates) were grown for 24 h in microtitre plates. They were washed and treated for 24 h with different concentrations of linezolid, tetracycline, rifampicin and vancomycin and four different SAMPs. After treatment, the redox indicator Alamar Blue was used to quantify metabolic activity of bacteria in biofilms, and confocal laser scanning microscopy with LIVE/DEAD staining was used to further elucidate any effects. RESULTS At MIC levels, rifampicin and tetracycline showed a marked reduction of metabolic activity in the S. epidermidis and S. haemolyticus biofilm. Linezolid had a moderate effect and vancomycin had a poor effect. MIC x10 and MIC x100 improved the antimicrobial activity of all antibiotics, especially vancomycin. However, metabolic activity was not completely suppressed in strong biofilm-producing strains. At MIC x10, the three most effective SAMPs (Ltx5, Ltx9 and Ltx10) were able to completely eliminate metabolic activity in the S. epidermidis and S. haemolyticus biofilms, which was also confirmed by complete cell death using confocal laser scanning microscopy investigations. Although none of the Ltx SAMPs could fully suppress metabolic activity in the S. aureus biofilm, their effect was superior to all tested antibiotics. CONCLUSIONS SAMPs had superior antimicrobial activity in staphylococcal biofilms compared with conventional antibiotics and are potential new therapeutic agents for biofilm-associated infections.

Conditional expression of retrovirally delivered anti-MYCN shRNA as an in vitro model system to study neuronal differentiation in MYCN-amplified neuroblastoma

BackgroundNeuroblastoma is a childhood cancer derived from immature cells of the sympathetic nervous system. The disease is clinically heterogeneous, ranging from neuronal differentiated benign ganglioneuromas to aggressive metastatic tumours with poor prognosis. Amplification of the MYCN oncogene is a well established poor prognostic factor found in up to 40% of high risk neuroblastomas.Using neuroblastoma cell lines to study neuronal differentiation in vitro is now well established. Several protocols, including exposure to various agents and growth factors, will differentiate neuroblastoma cell lines into neuron-like cells. These cells are characterized by a neuronal morphology with long extensively branched neurites and expression of several neurospecific markers.ResultsIn this study we use retrovirally delivered inducible short-hairpin RNA (shRNA) modules to knock down MYCN expression in MYCN-amplified (MNA) neuroblastoma cell lines. By addition of the inducer doxycycline, we show that the Kelly and SK-N-BE(2) neuroblastoma cell lines efficiently differentiate into neuron-like cells with an extensive network of neurites. These cells are further characterized by increased expression of the neuronal differentiation markers NFL and GAP43. In addition, we show that induced expression of retrovirally delivered anti-MYCN shRNA inhibits cell proliferation by increasing the fraction of MNA neuroblastoma cells in the G1 phase of the cell cycle and that the clonogenic growth potential of these cells was also dramatically reduced.ConclusionWe have developed an efficient MYCN-knockdown in vitro model system to study neuronal differentiation in MNA neuroblastomas.

Age-dependent prevalence of BK virus IgG and IgM antibodies measured by enzyme-linked immunosorbent assays (ELISA).

Enzyme immunoassays (ELISA) have been developed for the detection of BK virus IgG- and IgM-antibodies. Specific IgG is detected by an antigen-coated solid phase test; IgM by an antibody capture method. These methods have been used to study the age-distribution of BK virus antibodies in Tromsø county in Northern Norway. The serum panels tested were: 60 sera from paediatric patients aged 0-1 year; 220 sera from healthy persons aged 1-82 years; 74 sera from healthy blood donors; 107 sera from healthy pregnant women. The age-distribution of BKV-IgG antibodies showed that primary infections took place predominantly between the ages of 1 and 6 years, and that there were no sex differences, either in the age-specific prevalence or in the level of BKV-IgG. We found no significant differences in the prevalence of BKV-IgM antibodies in healthy children and adults and pregnant women. BKV-IgM was detected in 26 of the 461 sera tested (5.6%).