1. |
- Chaudhari, Aditi, et al.
(author)
-
p110alpha hot spot mutations E545K and H1047R exert metabolic reprogramming independently of p110alpha kinase activity : Kinase-independent signaling of p110 alpha mutants
- 2015
-
In: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 35:19, s. 3258-3273
-
Journal article (peer-reviewed)abstract
- The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit p110α is the most frequently mutated kinase in human cancer, and the hot spot mutations E542K, E545K, and H1047R are the most common mutations in p110α. Very little is known about the metabolic consequences of the hot spot mutations of p110α in vivo. In this study, we used adenoviral gene transfer in mice to investigate the effects of the E545K and H1047R mutations on hepatic and whole-body glucose metabolism. We show that hepatic expression of these hot spot mutations results in rapid hepatic steatosis, paradoxically accompanied by increased glucose tolerance, and marked glycogen accumulation. In contrast, wild-type p110α expression does not lead to hepatic accumulation of lipids or glycogen despite similar degrees of upregulated glycolysis and expression of lipogenic genes. The reprogrammed metabolism of the E545K and H1047R p110α mutants was surprisingly not dependent on altered p110α lipid kinase activity.
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2. |
- Abel, Frida, 1974, et al.
(author)
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Analyses of apoptotic regulators CASP9 and DFFA at 1P36.2, reveal rare allele variants in human neuroblastoma tumours.
- 2002
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In: British journal of cancer. - : Springer Science and Business Media LLC. - 0007-0920 .- 1532-1827. ; 86:4, s. 596-604
-
Journal article (peer-reviewed)abstract
- The genes encoding Caspase-9 and DFF45 have both recently been mapped to chromosome region 1p36.2, that is a region alleged to involve one or several tumour suppressor genes in neuroblastoma tumours. This study presents an update contig of the 'Smallest Region of Overlap of deletions' in Scandinavian neuroblastoma tumours and suggests that DFF45 is localized in the region. The genomic organization of the human DFF45 gene, deduced by in-silico comparisons of DNA sequences, is described for the first time in this paper. In the present study 44 primary tumours were screened for mutation by analysis of the genomic sequences of the genes. In two out of the 44 tumours this detected in the DFFA gene one rare allele variant that caused a non-polar to a polar amino acid exchange in a preserved hydrophobic patch of DFF45. One case was hemizygous due to deletion of the more common allele of this polymorphism. Out of 194 normal control alleles only one was found to carry this variant allele, so in respect of it, no healthy control individual out of 97 was homozygous. Moreover, our RT-PCR expression studies showed that DFF45 is preferably expressed in low-stage neuroblastoma tumours and to a lesser degree in high-stage neuroblastomas. We conclude that although coding mutations of Caspase-9 and DFF45 are infrequent in neuroblastoma tumours, our discovery of a rare allele in two neuroblastoma cases should be taken to warrant further studies of the role of DFF45 in neuroblastoma genetics.
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3. |
- Abel, Frida, 1974, et al.
(author)
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Gain of chromosome arm 17q is associated with unfavourable prognosis in neuroblastoma, but does not involve mutations in the somatostatin receptor 2(SSTR2) gene at 17q24.
- 1999
-
In: British journal of cancer. - : Springer Science and Business Media LLC. - 0007-0920 .- 1532-1827. ; 81:8, s. 1402-9
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Journal article (peer-reviewed)abstract
- Deletion of chromosome arm 1p and amplification of the MYCN oncogene are well-recognized genetic alterations in neuroblastoma cells. Recently, another alteration has been reported; gain of the distal part of chromosome arm 17q. In this study 48 neuroblastoma tumours were successfully analysed for 17q status in relation to known genetic alterations. Chromosome 17 status was detected by fluorescence in situ hybridization (FISH). Thirty-one of the 48 neuroblastomas (65%) showed 17q gain, and this was significantly associated with poor prognosis. As previously reported, 17q gain was significantly associated with metastatic stage 4 neuroblastoma and more frequently detected than both deletion of chromosome arm 1p and MYCN amplification in tumours of all stages. 17q gain also showed a strong correlation to survival probability (P = 0.0009). However, the most significant correlation between 17q gain and survival probability was observed in children with low-stage tumours (stage 1, 2, 3 and 4S), with a survival probability of 100% at 5 years from diagnosis for children with tumours showing no 17q gain compared to 52.5% for those showing 17q gain (P = 0.0021). This suggests that 17q gain as a prognostic factor plays a more crucial role in low-stage tumours. Expression of the somatostatin receptor 2 (SSTR2), localized in chromosome region 17q24, has in previous studies been shown to be positively related to survival in neuroblastoma. A point mutation in the SSTR2 gene has earlier been reported in a human small-cell lung cancer. In this study, mutation screening of the SSTR2 gene in 43 neuroblastoma tumours was carried out with polymerase chain reaction-based single-stranded conformation polymorphism/heteroduplex (SSCP/HD) and DNA sequencing, and none of the tumours showed any aberrations in the SSTR2 gene. These data suggest that mutations in the SSTR2 gene are uncommon in neuroblastoma tumours and do not correlate with either the 17q gain often seen or the reason some tumours do not express SSTR2 receptors. Overall, this study indicates that gain of chromosome arm 17q is the most frequently occurring genetic alteration, and that it is associated with established prognostic factors.
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4. |
- Carén, Helena, 1979, et al.
(author)
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A cluster of genes located in 1p36 are down-regulated in neuroblastomas with poor prognosis, but not due to CpG island methylation.
- 2005
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In: Molecular cancer. - : Springer Science and Business Media LLC. - 1476-4598. ; 4:1
-
Journal article (peer-reviewed)abstract
- BACKGROUND: A common feature of neuroblastoma tumours are partial deletions of the short arm of chromosome 1 (1p-deletions). This is indicative of a neuroblastoma tumour suppressor gene being located in the region. Several groups including our have been studying candidate neuroblastoma genes in the region, but no gene/genes have yet been found that fulfil the criteria for being a neuroblastoma tumour suppressor. Since frequent mutations have not been detected, we have now analyzed the expression and promoter CpG island methylation status of the genes UBE4B, KIF1B, PGD, APITD1, DFFA and PEX14 in the 1p36.22 region in order to find an explanation for a possible down-regulation of this region. RESULTS: The current study shows that gene transcripts in high stage neuroblastoma tumours are significantly down-regulated compared to those in low stage tumours in the 1p36.22 region. CpG island methylation does not seem to be the mechanism of down-regulation for most of the genes tested, since no methylation was detected in the fragments analyzed. One exception is the CpG island of APITD1. Methylation of this gene is also seen in blood from control individuals and is therefore not believed to participate in tumour development. CONCLUSION: The genes UBE4B, KIF1B, PGD, APITD1, DFFA and PEX14 are down-regulated in high stage NB tumours, a feature that can not be explained by CpG island methylation.
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5. |
- Carén, Helena, 1979, et al.
(author)
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Genetic and epigenetic changes in the common 1p36 deletion in neuroblastoma tumours.
- 2007
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In: British Journal of Cancer. - : Springer Science and Business Media LLC. - 0007-0920 .- 1532-1827.
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Journal article (peer-reviewed)abstract
- Chromosome 1p is frequently deleted in neuroblastoma (NB) tumours. The commonly deleted region has been narrowed down by loss of heterozygosity studies undertaken by different groups. Based on earlier mapping data, we have focused on a region on 1p36 (chr1: 7 765 595-11 019 814) and performed an analysis of 30 genes by exploring features such as epigenetic regulation, that is DNA methylation and histone deacetylation, mutations at the DNA level and mRNA expression. Treatment of NB cell lines with the histone deacetylase inhibitor trichostatin A led to increased gene transcription of four of the 30 genes, ERRFI1 (MIG-6), PIK3CD, RBP7 (CRBPIV) and CASZ1, indicating that these genes could be affected by epigenetic downregulation in NBs. Two patients with nonsynonymous mutations in the PIK3CD gene were detected. One patient harboured three variations in the same exon, and p.R188W. The other patient had the variation p.M655I. In addition, synonymous variations and one variation in an intronic sequence were also found. The mRNA expression of this gene is downregulated in unfavourable, compared to favourable, NBs. One nonsynonymous mutation was also identified in the ERRFI1 gene, p.N343S, and one synonymous. None of the variations above were found in healthy control individuals. In conclusion, of the 30 genes analysed, the PIK3CD gene stands out as one of the most interesting for further studies of NB development and progression.British Journal of Cancer advance online publication, 16 October 2007; doi:10.1038/sj.bjc.6604032 www.bjcancer.com.
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6. |
- Chaudhari, Aditi, et al.
(author)
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Hepatic deletion of p110α and p85α results in insulin resistance despite sustained IRS1-associated phosphatidylinositol kinase activity
- 2017
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In: F1000 Research. - : Faculty of 1000 Ltd.. - 2046-1402 .- 1759-796X. ; 6
-
Journal article (peer-reviewed)abstract
- Background: Class IA phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) is an integral mediator of insulin signaling. The p110 catalytic and p85 regulatory subunits of PI3K are the products of separate genes, and while they come together to make the active heterodimer, they have opposing roles in insulin signaling and action. Deletion of hepatic p110α results in an impaired insulin signal and severe insulin resistance, whereas deletion of hepatic p85α results in improved insulin sensitivity due to sustained levels of phosphatidylinositol (3,4,5)-trisphosphate. Here, we created mice with combined hepatic deletion of p110α and p85α (L-DKO) to study the impact on insulin signaling and whole body glucose homeostasis.Methods: Six-week old male flox control and L-DKO mice were studied over a period of 18 weeks, during which weight and glucose levels were monitored, and glucose tolerance tests, insulin tolerance test and pyruvate tolerance test were performed. Fasting insulin, insulin signaling mediators, PI3K activity and insulin receptor substrate (IRS)1-associated phosphatidylinositol kinase activity were examined at 10 weeks. Liver, muscle and white adipose tissue weight was recorded at 10 weeks and 25 weeks.Results: The L-DKO mice showed a blunted insulin signal downstream of PI3K, developed markedly impaired glucose tolerance, hyperinsulinemia and had decreased liver and adipose tissue weights. Surprisingly, however, these mice displayed normal hepatic glucose production, normal insulin tolerance, and intact IRS1-associated phosphatidylinositol kinase activity without compensatory upregulated signaling of other classes of PI3K.Conclusions: The data demonstrate an unexpectedly overall mild metabolic phenotype of the L-DKO mice, suggesting that lipid kinases other than PI3Ks might partially compensate for the loss of p110α/p85α by signaling through other nodes than Akt/Protein Kinase B.
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7. |
- Chaudhari, Aditi, et al.
(author)
-
p110α hot spot mutations E545K and H1047R exert metabolic reprogramming independently of p110α kinase activity
- 2015
-
In: Molecular and Cellular Biology. - : American Society for Microbiology. - 0270-7306 .- 1098-5549. ; 35:19, s. 3258-3273
-
Journal article (peer-reviewed)abstract
- The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit p110α is the most frequently mutated kinase in human cancer, and the hot spot mutations E542K, E545K, and H1047R are the most common mutations in p110α. Very little is known about the metabolic consequences of the hot spot mutations of p110α in vivo. In this study, we used adenoviral gene transfer in mice to investigate the effects of the E545K and H1047R mutations on hepatic and whole-body glucose metabolism. We show that hepatic expression of these hot spot mutations results in rapid hepatic steatosis, paradoxically accompanied by increased glucose tolerance, and marked glycogen accumulation. In contrast, wild-type p110α expression does not lead to hepatic accumulation of lipids or glycogen despite similar degrees of upregulated glycolysis and expression of lipogenic genes. The reprogrammed metabolism of the E545K and H1047R p110α mutants was surprisingly not dependent on altered p110α lipid kinase activity.
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8. |
- Dahl-Halvarsson, Martin, et al.
(author)
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Drosophila model of myosin myopathy rescued by overexpression of a TRIM-protein family member
- 2018
-
In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 115:28
-
Journal article (peer-reviewed)abstract
- Myosin is a molecular motor indispensable for body movement and heart contractility. Apart from pure cardiomyopathy, mutations in MYH7 encoding slow/beta-cardiac myosin heavy chain also cause skeletal muscle disease with or without cardiac involvement. Mutations within the alpha-helical rod domain of MYH7 are mainly associated with Laing distal myopathy. To investigate the mechanisms underlying the pathology of the recurrent causative MYH7 mutation (K1729del), we have developed a Drosophila melanogaster model of Laing distal myopathy by genomic engineering of the Drosophila Mhc locus. Homozygous MhcK1728del animals die during larval/pupal stages, and both homozygous and heterozygous larvae display reduced muscle function. Flies expressing only MhcK1728del in indirect flight and jump muscles, and heterozygous MhcK1728del animals, were flightless, with reduced movement and decreased lifespan. Sarcomeres of MhcK1728del mutant indirect flight muscles and larval body wall muscles were disrupted with clearly disorganized muscle filaments. Homozygous MhcK1728del larvae also demonstrated structural and functional impairments in heart muscle, which were not observed in heterozygous animals, indicating a dose-dependent effect of the mutated allele. The impaired jump and flight ability and the myopathy of indirect flight and leg muscles associated with MhcK1728del were fully suppressed by expression of Abba/Thin, an E3-ligase that is essential for maintaining sarcomere integrity. This model of Laing distal myopathy in Drosophila recapitulates certain morphological phenotypic features seen in Laing distal myopathy patients with the recurrent K1729del mutation. Our observations that Abba/Thin modulates these phenotypes suggest that manipulation of Abba/Thin activity levels may be beneficial in Laing distal myopathy.
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9. |
- Dahl-Halvarsson, Martin, et al.
(author)
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Impaired muscle morphology in a Drosophila model of myosin storage myopathy was supressed by overexpression of an E3 ubiquitin ligase
- 2020
-
In: Disease Models & Mechanisms. - : The Company of Biologists. - 1754-8403 .- 1754-8411. ; 13:12
-
Journal article (peer-reviewed)abstract
- Myosin is vital for body movement and heart contractility. Mutations in MYH7, encoding slow/beta-cardiac myosin heavy chain, are an important cause of hypertrophic and dilated cardiomyopathy, as well as skeletal muscle disease. A dominant missense mutation (R1845W) in MYH7 has been reported in several unrelated cases of myosin storage myopathy. We have developed a Drosophila model for a myosin storage myopathy in order to investigate the dose-dependent mechanisms underlying the pathological roles of the R1845W mutation. This study shows that a higher expression level of the mutated allele is concomitant with severe impairment of muscle function and progressively disrupted muscle morphology. The impaired muscle morphology associated with the mutant allele was suppressed by expression of Thin (herein referred to as Abba), an E3 ubiquitin ligase. This Drosophila model recapitulates pathological features seen in myopathy patients with the R1845W mutation and severe ultrastructural abnormalities, including extensive loss of thick filaments with selective A-band loss, and preservation of I-band and Z-disks were observed in indirect flight muscles of flies with exclusive expression of mutant myosin. Furthermore, the impaired muscle morphology associated with the mutant allele was suppressed by expression of Abba. These findings suggest that modification of the ubiquitin proteasome system may be beneficial in myosin storage myopathy by reducing the impact of MYH7 mutation in patients.
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10. |
- Deland, Lily, et al.
(author)
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Discovery of a rare GKAP1-NTRK2 fusion in a pediatric low-grade glioma, leading to targeted treatment with TRK-inhibitor larotrectinib
- 2021
-
In: Cancer Biology & Therapy. - : Taylor & Francis. - 1538-4047 .- 1555-8576. ; 22:3, s. 184-195
-
Journal article (peer-reviewed)abstract
- Here we report a case of an 11-year-old girl with an inoperable tumor in the optic chiasm/hypothalamus, who experienced several tumor progressions despite three lines of chemotherapy treatment. Routine clinical examination classified the tumor as a BRAF-negative pilocytic astrocytoma. Copy-number variation profiling of fresh frozen tumor material identified two duplications in 9q21.32–33 leading to breakpoints within the GKAP1 and NTRK2 genes. RT-PCR Sanger sequencing revealed a GKAP1-NTRK2 exon 10–16 in-frame fusion, generating a putative fusion protein of 658 amino acids with a retained tyrosine kinase (TK) domain. Functional analysis by transient transfection of HEK293 cells showed the GKAP1-NTRK2 fusion protein to be activated through phosphorylation of the TK domain (Tyr705). Subsequently, downstream mediators of the MAPK- and PI3K-signaling pathways were upregulated in GKAP1-NTRK2 cells compared to NTRK2 wild-type; phosphorylated (p)ERK (3.6-fold), pAKT (1.8- fold), and pS6 ribosomal protein (1.4-fold). Following these findings, the patient was enrolled in a clinical trial and treated with the specific TRK-inhibitor larotrectinib, resulting in the arrest of tumor growth. The patient’s condition is currently stable and the quality of life has improved significantly. Our findings highlight the value of comprehensive clinical molecular screening of BRAF-negative pediatric low-grade gliomas, to reveal rare fusions serving as targets for precision therapy.
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11. |
- Deland, Lily, et al.
(author)
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Novel TPR::ROS1 Fusion Gene Activates MAPK, PI3K and JAK/STAT Signaling in an Infant-type Pediatric Glioma.
- 2022
-
In: Cancer genomics & proteomics. - : Anticancer Research USA Inc.. - 1109-6535 .- 1790-6245. ; 19:6, s. 711-726
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Journal article (peer-reviewed)abstract
- Although fusion genes involving the proto-oncogene receptor tyrosine kinase ROS1 are rare in pediatric glioma, targeted therapies with small inhibitors are increasingly being approved for histology-agnostic fusion-positive solid tumors.Here, we present a 16-month-old boy, with a brain tumor in the third ventricle. The patient underwent complete resection but relapsed two years after diagnosis and underwent a second operation. The tumor was initially classified as a low-grade glioma (WHO grade 2); however, methylation profiling suggested the newly WHO-recognized type: infant-type hemispheric glioma. To further refine the molecular background, and search for druggable targets, whole genome (WGS) and whole transcriptome (RNA-Seq) sequencing was performed.Concomitant WGS and RNA-Seq analysis revealed several segmental gains and losses resulting in complex structural rearrangements and fusion genes. Among the top-candidates was a novel TPR::ROS1 fusion, for which only the 3' end of ROS1 was expressed in tumor tissue, indicating that wild type ROS1 is not normally expressed in the tissue of origin. Functional analysis by Western blot on protein lysates from transiently transfected HEK293 cells showed the TPR::ROS1 fusion gene to activate the MAPK-, PI3K- and JAK/STAT- pathways through increased phosphorylation of ERK, AKT, STAT and S6. The downstream pathway activation was also confirmed by immunohistochemistry on tumor tissue slides from the patient.We have mapped the activated oncogenic pathways of a novel ROS1-fusion gene and broadened the knowledge of the newly recognized infant-type glioma subtype. The finding facilitates suitable targeted therapies for the patient in case of relapse.
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12. |
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13. |
- Ejeskär, Katarina, 1969, et al.
(author)
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Fine mapping of a tumour suppressor candidate gene region in 1p36.2-3, commonly deleted in neuroblastomas and germ cell tumours.
- 2001
-
In: Medical and pediatric oncology. - 0098-1532 .- 1096-911X. ; 36:1, s. 61-6
-
Journal article (peer-reviewed)abstract
- BACKGROUND: A common genetic feature of neuroblastomas, which is also an important prognostic factor, is deletion of chromosome region 1p. The deletion of 1p often involves a deletion of varying size, with a consensus region within the most distal bands 1p36.2-3. The neuroblastoma SRO (shortest region of overlap of (deletions) presented earlier by our group was defined distally by the cluster of loci D1S80/ D1Z2/CDC2L1 and proximally by loci D1S244, i.e., approximately 25 cM. The 1p deletions are, however, not restricted to neuroblastoma tumours. In fact, a large spectrum of tumour types display deletions to varying degrees of 1p. PROCEDURE: We have exploited the possibility of using deletions of other tumour types, preferentially that of germ cell tumours, and combining the deletions with that of the neuroblastoma SRO. Also in germ cell tumours, distal 1p-deletions have been shown to have prognostic significance. RESULTS: We found in our germ cell tumours a SRO ranging from D1S508 to D1S200. Interestingly, this region only partially overlapped (approximately 5 cm) with our neuroblastoma SRO in region D1S508 to D1S244. We have thus focused on analysing this smaller region in the search for genes involved in the genesis of different cancers. We have performed radiation hybrid mapping of a large number of markers, STSs, ESTs, and others known to reside in 1p. We have also initiated the development of a BAC contig of the region. FISH, and fibre-FISH mapping of BACs were also performed. CONCLUSIONS: The data presented here constitute an ongoing work with the aim of identifying and cloning gene(s) important for development of germ cell tumours, neuroblastomas, and possibly other tumours.
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14. |
- Ejeskär, Katarina, 1969, et al.
(author)
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Fine mapping of the human preprocortistatin gene (CORT) to neuroblastoma consensus deletion region 1p36.3-->p36.2, but absence of mutations in primary tumors.
- 2000
-
In: Cytogenetics and cell genetics. - : S. Karger AG. - 0301-0171. ; 89:1-2, s. 62-6
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Journal article (peer-reviewed)abstract
- The processed product of the human gene preprocortistatin, the peptide cortistatin-17 (hCST-17), bears a strong structural resemblance to the peptide somatostatin (SST), which has an identical receptor binding domain. CST has affinity to all known SST receptor (SSTR) subtypes. Expression of both SST and its receptors has been shown in previous studies to have biological and clinical significance in neuroblastomas, with a putative role in tumor differentiation and apoptosis in vivo. In this work we have employed radiation hybrid mapping and BAC physical mapping to map the human preprocortistatin gene (CORT) to chromosome region 1p36.3-->p36.2, close to the genetic marker D1S244. D1S244 defines the centromeric border of the smallest region of overlap of deletion in our primary neuroblastoma material. We have also defined the genomic sequence of the gene by BAC sequencing and found that preprocortistatin consists of two exons divided by a 1-kb intron. Two polymorphic sites, neither of which causes amino acid exchange, have been detected in the coding region of the gene. Expression studies showed that preprocortistatin is expressed in neuroblastomas of all different stages, as well as in ganglioneuromas. Through genomic sequencing we made mutation analyses of exonic sequences in 49 primary neuroblastomas of all different stages, but no mutations could be detected.
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15. |
- Ejeskär, Katarina, 1969
(author)
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Genetic alterations in Scandinavian neuroblastoma tumors
- 2000
-
Doctoral thesis (other academic/artistic)abstract
- The results presented here derive from our efforts to find genes and chromosomal regions of interest in the formation and progression of the childhood tumor neuroblastoma.Neuroblastoma is the most common extracranial solid tumor of childhood. It is a tumor of the postganglionic sympathetic nervous system, and clinically, the hallmark of neuroblastoma is its heterogeneity.In order to characterize chromosomal regions of interest the methods loss of heterozygozity (LOH) studies using PCR-based polymorphic markers, representational difference analysis (RDA), somatic cell hybrid mapping, fluorescent in situ hybridization (FISH), radiation hybrid mapping and physical mapping by construction of a BAC-contig have been used. For mutation analysis of genes the methods single strand conformation polymorphism (SSCP), heteroduplex (HD) and DNA-sequencing have been used, and the expression studies of candidate genes have been performed using RT-PCR.In the Scandinavian neuroblastoma tumor material we have been able to show that deletions of chromosome arm 3p is the second most common deleted chromosomal region (16%) next to 1p (22%). We could also see a difference in clinical outcome between patients with tumors displaying deletion of the entire chromosome 3 versus the ones with tumors displaying deletions of region 3p only. The ones with 3p-deletion have all died of disease whereas the ones with entire chromosome 3 loss are alive and well. We have also characterized a region on chromosome 1, 1p36, shown, by others and us, to be commonly deleted in neuroblastoma tumors. A critical region of app. 25 cM between genetic markers D1S80 and D1S244 could be defined. We have also by using combined LOH-data from both neuroblastoma and germ cell tumors defined a region of common deletion for both tumor types, this region could be defined by markers D1S508 and D1S244 and is app. 5 cM.Also some 1p36 putative tumor suppressor genes, i.e. CDC2L1, TP73 and CORT, have been fine mapped and tested for expression and mutations in neuroblastomas. No evidence for any of them to be the 1p36 neuroblastoma tumor suppressor gene has however been discovered.
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16. |
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17. |
- Ejeskär, Katarina, 1969, et al.
(author)
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Introduction of in vitro transcribed ENO1 mRNA into neuroblastoma cells induces cell death.
- 2005
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In: BMC cancer. - : Springer Science and Business Media LLC. - 1471-2407. ; 5
-
Journal article (peer-reviewed)abstract
- BACKGROUND: Neuroblastoma is a solid tumour of childhood often with an unfavourable outcome. One common genetic feature in aggressive tumours is 1p-deletion.The alpha-enolase (ENO1) gene is located in chromosome region 1p36.2, within the common region of deletion in neuroblastoma. One alternative translated product of the ENO1 gene, known as MBP-1, acts as a negative regulator of the c-myc oncogene, making the ENO1 gene a candidate as a tumour suppressor gene. METHODS: Methods used in this study are transfection of cDNA-vectors and in vitro transcribed mRNA, cell growth assay, TUNEL-assay, real-time RT-PCR (TaqMan) for expression studies, genomic sequencing and DHPLC for mutation detection. RESULTS: Here we demonstrate that transfection of ENO1 cDNA into 1p-deleted neuroblastoma cell lines causes' reduced number of viable cells over time compared to a negative control and that it induces apoptosis. Interestingly, a similar but much stronger dose-dependent reduction of cell growth was observed by transfection of in vitro transcribed ENO1 mRNA into neuroblastoma cells. These effects could also be shown in non-neuroblastoma cells (293-cells), indicating ENO1 to have general tumour suppressor activity.Expression of ENO1 is detectable in primary neuroblastomas of all different stages and no difference in the level of expression can be detected between 1p-deleted and 1p-intact tumour samples. Although small numbers (11 primary neuroblastomas), there is some evidence that Stage 4 tumours has a lower level of ENO1-mRNA than Stage 2 tumours (p = 0.01). However, mutation screening of 44 primary neuroblastomas of all different stages, failed to detect any mutations. CONCLUSION: Our studies indicate that ENO1 has tumour suppressor activity and that high level of ENO1 expression has growth inhibitory effects.
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18. |
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19. |
- Ejeskär, Katarina, 1969, et al.
(author)
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Method for efficient transfection of in vitro-transcribed mRNA into SK-N-AS and HEK293 cells: difference in the toxicity of nuclear EGFP compared to cytoplasmic EGFP.
- 2006
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In: International journal of molecular medicine. - 1107-3756. ; 17:6, s. 1011-6
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Journal article (peer-reviewed)abstract
- Here we report a method for efficient transfection of in vitro-transcribed mRNA into two different types of human adherent cells, the neuroblastoma cell line SK-N-AS, and the transformed kidney cell line HEK293. By using newly trypsinized adherent cells in suspension and Lipofectaminetrade mark 2000, we detected a transfection efficiency of 80-90% in both cell lines and a cell viability of 90% in SK-N-AS and 60% in HEK293, 24 h after transfection when using cytoplasmic enhanced green fluorescent protein (EGFP)-mRNA. We have evaluated the different effects of the generally used EGFP that mainly localizes to the cytoplasm and nuclear EGFP, where the nuclear EGFP are more toxic to the cells than the cytoplasmic EGFP. In order to develop a null experiment, we constructed a short non-functional mRNA including a nuclear localization signal and evaluated the concentrations at which mRNA encoding nuclear proteins can be added without a general toxicity, depending on the fact that the proteins are localized to the nucleus. For both SK-N-AS and HEK293 cells, a concentration of up to 100 ng mRNA in 10(5) cells, encoding a nuclear protein with no other function, did not affect the cells. For evaluation of the method, we screened four different human mRNAs, PDG, DFFA, CORT and PEX14, for their ability to affect cell proliferation in these cells. PEX14 was the only gene that significantly (p=0.03) reduced cell proliferation for both cell types, DFFA significantly (p=0.04) reduced cell proliferation in SK-N-AS but not in HEK293 cells. PGD and CORT did not have any effect on cell proliferation. We have developed an easy method for efficient delivery of in vitro-transcribed mRNA into the adherent cell lines, SK-N-AS and HEK293. This method is useful for a quick screening of how different genes affect cell proliferation.
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20. |
- Ejeskär, Katarina, 1969, et al.
(author)
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The Unique Non-Catalytic C-Terminus of P37delta-PI3K Adds Proliferative Properties In Vitro and In Vivo
- 2015
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In: Plos One. - : Public Library of Science (PLoS). - 1932-6203. ; 10:5
-
Journal article (peer-reviewed)abstract
- The PI3K/Akt pathway is central for numerous cellular functions and is frequently deregulated in human cancers. The catalytic subunits of PI3K, p110, are thought to have a potential oncogenic function, and the regulatory subunit p85 exerts tumor suppressor properties. The fruit fly, Drosophila melanogaster, is a highly suitable system to investigate PI3K signaling, expressing one catalytic, Dp110, and one regulatory subunit, Dp60, and both show strong homology with the human PI3K proteins p110 and p85. We recently showed that p37 delta, an alternatively spliced product of human PI3K p110 delta, displayed strong proliferation-promoting properties despite lacking the catalytic domain completely. Here we functionally evaluate the different domains of human p37 delta in Drosophila. The N-terminal region of Dp110 alone promotes cell proliferation, and we show that the unique C-terminal region of human p37 delta further enhances these proliferative properties, both when expressed in Drosophila, and in human HEK-293 cells. Surprisingly, although the N-terminal region of Dp110 and the C-terminal region of p37 delta both display proliferative effects, over-expression of full length Dp110 or the N-terminal part of Dp110 decreases survival in Drosophila, whereas the unique C-terminal region of p37 delta prevents this effect. Furthermore, we found that the N-terminal region of the catalytic subunit of PI3K p110, including only the Dp60 (p85)-binding domain and a minor part of the Ras binding domain, rescues phenotypes with severely impaired development caused by Dp60 over-expression in Drosophila, possibly by regulating the levels of Dp60, and also by increasing the levels of phosphorylated Akt. Our results indicate a novel kinase-independent function of the PI3K catalytic subunit.
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21. |
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22. |
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23. |
- Fransson, Susanne, 1975, et al.
(author)
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Neuroblastoma tumors with favorable and unfavorable outcomes: Significant differences in mRNA expression of genes mapped at 1p36.2.
- 2007
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In: Genes, chromosomes & cancer. - : Wiley. - 1045-2257 .- 1098-2264. ; 46:1, s. 45-52
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Journal article (peer-reviewed)abstract
- The distal part of 1p is frequently deleted in aggressive neuroblastoma, and the region is believed to harbor one or more tumor suppressor genes relevant to tumor development. To analyze differences among neuroblastoma tumors, an expression profile was established for the genes mapped within a previously described shortest region of overlap of deletions at 1p36.2. The gene expression levels were quantified by TaqMan real-time (RT)-PCR for 30 transcripts using 55 primary neuroblastoma tumors. Here we report on a significant decrease in gene expression of the genes RERE, PIK3CD, LZIC, PGD, and PEX14 and an increase of SLC2A5 when comparing tumors of favorable biology to Stage 4 neuroblastomas. When comparing 1p-deleted tumors of all stages to tumors with an intact 1p, a significant difference at gene-by-gene level in TNFRSF9, RERE, PIK3CD, CLSTN1, CTNNBIP1, and CASZ1 was detected. A complete loss of expression could not be seen for any single gene analyzed. Several of the genes with diminished expression in unfavorable or 1p-deleted tumors have functions that could contribute to tumor development. It is also possible that a combination of lowly expressed genes at 1p, rather than one single classical tumor suppressor gene, causes the unfavorable outcome associated with 1p-deletion in neuroblastoma.
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- Jurcevic, Sanja, 1971-, et al.
(author)
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Bioinformatics analysis of miRNAs in the neuroblastoma 11q-deleted region reveals a role of miR-548l in both 11q-deleted and MYCN amplified tumour cells
- 2022
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In: Scientific Reports. - : Springer Nature. - 2045-2322. ; 12:1
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Journal article (peer-reviewed)abstract
- Neuroblastoma is a childhood tumour that is responsible for approximately 15% of all childhood cancer deaths. Neuroblastoma tumours with amplification of the oncogene MYCN are aggressive, however, another aggressive subgroup without MYCN amplification also exists; rather, they have a deleted region at chromosome arm 11q. Twenty-six miRNAs are located within the breakpoint region of chromosome 11q and have been checked for a possible involvement in development of neuroblastoma due to the genomic alteration. Target genes of these miRNAs are involved in pathways associated with cancer, including proliferation, apoptosis and DNA repair. We could show that miR-548l found within the 11q region is downregulated in neuroblastoma cell lines with 11q deletion or MYCN amplification. In addition, we showed that the restoration of miR-548l level in a neuroblastoma cell line led to a decreased proliferation of these cells as well as a decrease in the percentage of cells in the S phase. We also found that miR-548l overexpression suppressed cell viability and promoted apoptosis, while miR-548l knockdown promoted cell viability and inhibited apoptosis in neuroblastoma cells. Our results indicate that 11q-deleted neuroblastoma and MYCN amplified neuroblastoma coalesce by downregulating miR-548l.
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