| 1. |
|
|
| 2. |
- Lennartsson, Andreas, et al.
(författare)
-
A murine antibacterial orthologue to human bactericidal/permeability-increasing protein (BPI) is expressed in testis, epididymis, and bone marrow.
- 2005
-
Ingår i: Journal of Leukocyte Biology. - : Oxford University Press (OUP). - 1938-3673 .- 0741-5400. ; 77:3, s. 369-377
-
Tidskriftsartikel (refereegranskat)abstract
- The bactericidal/permeability-increasing protein (BPI), stored in human neutrophil granulocytes, is cytotoxic against Gram-negative bacteria. Several genes related to BPI cluster on human chromosome 20 and on mouse chromosome 2, but expression and characterization of a BPI ortholog in the mouse have not been reported. We asked whether BPI is structurally and functionally conserved between humans and mice and whether murine BPI might be synthesized in neutrophils as well as in other tissues. We report the isolation of a murine full-length cDNA encoding a 54-kDa protein, showing 53% amino acid identity and 71% similarity, to human BPI. The murine BPI and human BPI genes show a similar exon-intron organization. Murine BPI mRNA was detected in testis, epididymis, and bone marrow, as well as in Sertoli and promyelocytic cell lines. Although levels of BPI mRNA in human and murine testis were comparable, expression in murine bone marrow cells was low as compared with that in human bone marrow. BPI protein showed a cytoplasmic, granular localization in mature neutrophils. BPI gene expression in Sertoli and promyelocytic cells was enhanced several-fold by all-trans retinoic acid. Overexpression of murine BPI in human embryonic kidney 293 cells resulted in antibacterial activity against Escherichia coli, comparable with that obtained with human BPI. In conclusion, it was demonstrated that mouse neutrophils store BPI with antibacterial activity and that murine BPI is also expressed in testis and epididymis.
|
|
| 3. |
- Lennartsson, Andreas, et al.
(författare)
-
All-trans retinoic acid-induced expression of bactericidal/permeability-increasing protein (BPI) in human myeloid cells correlates to binding of C/EBP{beta} and C/EBP{varepsilon} to the BPI promoter.
- 2006
-
Ingår i: Journal of Leukocyte Biology. - : Oxford University Press (OUP). - 1938-3673 .- 0741-5400. ; 80:1, s. 196-203
-
Tidskriftsartikel (refereegranskat)abstract
- Bactericidal/permeability-inereasing protein (BPI) neutralizes the proinflammatory effects of lipopolysaccharide and is of potential clinical use in the treatment of fulminant Gram-negative infections. BPI is a cationic protein with antibacterial activity stored in azurophil (primary) granules of neutrophil granulocytes. However, the absence of BPI in patients with specific granule deficiency indicates a transcriptional control of BPI, which is distinct from that of other azurophil granule proteins. Accordingly, we demonstrate in vivo that the BPI mRNA level peaks, together with mRNA for specific granule proteins, during the myelocytic and metamyelocytic stage of granulocytic maturation. The human promyelocytic cell line NB4 expresses several azurophil granule proteins, but expression of BPI is undetectable. We show that treatment of NB4 cells with all-trans retinoic acid (ATRA) induces BPI expression at mRNA and at protein level. The induction is dependent on de novo protein synthesis, as judged by sensitivity to cycloheximide. Previous investigations have indicated a potential role of CCAAT/enhancer-binding protein (C/EBP) transcription factors in the regulation of BPI expression. Here, we show that induction of NB4 cells with ATRA correlates to direct binding of C/EBP beta and C/EBP epsilon to the proximal BPI promoter, as determined by electrophoretic mobility shift analysis and chromatin immunoprecipitation. The dependency on C/EBP beta and C/EBP epsilon provides an explanation for delayed BPI mRNA expression, as compared with mRNA of other azurophil granule proteins.
|
|
| 4. |
|
|
| 5. |
- Montano, Giorgia, et al.
(författare)
-
Role of WT1-ZNF224 interaction in the expression of apoptosis-regulating genes
- 2013
-
Ingår i: Human Molecular Genetics. - : Oxford University Press (OUP). - 0964-6906 .- 1460-2083. ; 22:9, s. 1771-1782
-
Tidskriftsartikel (refereegranskat)abstract
- The transcription factor Wilms tumor gene 1, WT1, is implicated both in normal developmental processes and in the generation of a variety of solid tumors and hematological malignancies. Physical interactions of other cellular proteins with WT1 are known to modulate its function. We previously identified the Krppel-like zinc-finger protein, ZNF224, as a novel human WT1-associating protein that enhances the transcriptional activation of the human vitamin D receptor promoter by WT1. Here, we have analyzed the effects of WT1ZNF224 interaction on the expression of apoptosis-regulating genes in the chronic myelogenous leukemia (CML) K562 cell line. The results demonstrated that ZNF224 acts in fine tuning of WT1-dependent control of gene expression, acting as a co-activator of WT1 in the regulation of proapoptotic genes and suppressing WT1 mediated transactivation of antiapoptotitc genes. Moreover, the DNA damaging drug cytosine arabinoside (ara-C) induces expression of ZNF224 in K562 cells and this induction enhances cell apoptotic response to ara-C. These findings suggest that ZNF224 can be a mediator of DNA damage-induced apoptosis in leukemia cells.
|
|
| 6. |
|
|
| 7. |
- Montano, Giorgia, et al.
(författare)
-
The hematopoietic tumor suppressor interferon regulatory factor 8 (IRF8) is upregulated by the antimetabolite cytarabine in leukemic cells involving the zinc finger protein ZNF224, acting as a cofactor of the Wilms' tumor gene 1 (WT1) protein.
- 2016
-
Ingår i: Leukemia Research: A Forum for Studies on Leukemia and Normal Hemopoiesis. - : Elsevier BV. - 1873-5835. ; 40:1, s. 60-67
-
Tidskriftsartikel (refereegranskat)abstract
- The transcription factor interferon regulatory factor-8 (IRF8) is highly expressed in myeloid progenitors, while most myeloid leukemias show low or absent expression. Loss of IRF8 in mice leads to a myeloproliferative disorder, indicating a tumor-suppressive role of IRF8. The Wilms tumor gene 1 (WT1) protein represses the IRF8-promoter. The zinc finger protein ZNF224 can act as a transcriptional co-factor of WT1 and potentiate the cytotoxic response to the cytostatic drug cytarabine. We hypothesized that cytarabine upregulates IRF8 and that transcriptional control of IRF8 involves WT1 and ZNF224. Treatment of leukemic K562 cells with cytarabine upregulated IRF8 protein and mRNA, which was correlated to increased expression of ZNF224. Knock down of ZNF224 with shRNA suppressed both basal and cytarabine-induced IRF8 expression. While ZNF224 alone did not affect IRF8 promoter activity, ZNF224 partially reversed the suppressive effect of WT1 on the IRF8 promoter, as judged by luciferase reporter experiments. Coprecipitation revealed nuclear binding of WT1 and ZNF224, and by chromatin immunoprecipitation (ChIP) experiments it was demonstrated that WT1 recruits ZNF224 to the IRF8 promoter. We conclude that cytarabine-induced upregulation of the IRF8 in leukemic cells involves increased levels of ZNF224, which can counteract the repressive activity of WT1 on the IRF8-promoter.
|
|
| 8. |
- Montano, Giorgia, et al.
(författare)
-
WT1-mediated repression of the proapoptotic transcription factor ZNF224 is triggered by the BCR-ABL oncogene.
- 2015
-
Ingår i: Oncotarget. - : Impact Journals, LLC. - 1949-2553. ; 29:6, s. 37-28223
-
Tidskriftsartikel (refereegranskat)abstract
- The Kruppel-like protein ZNF224 is a co-factor of the Wilms' tumor 1 protein, WT1. We have previously shown that ZNF224 exerts a specific proapoptotic role in chronic myelogenous leukemia (CML) K562 cells and contributes to cytosine arabinoside-induced apoptosis, by modulating WT1-dependent transcription of apoptotic genes. Here we demonstrate that ZNF224 gene expression is down-regulated both in BCR-ABL positive cell lines and in primary CML samples and is restored after imatinib and second generation tyrosine kinase inhibitors treatment. We also show that WT1, whose expression is positively regulated by BCR-ABL, represses transcription of the ZNF224 gene. Finally, we report that ZNF224 is significantly down-regulated in patients with BCR-ABL positive chronic phase-CML showing poor response or resistance to imatinib treatment as compared to high-responder patients. Taken as a whole, our data disclose a novel pathway activated by BCR-ABL that leads to inhibition of apoptosis through the ZNF224 repression. ZNF224 could thus represent a novel promising therapeutic target in CML.
|
|
| 9. |
|
|
| 10. |
- Möller, Mattias, et al.
(författare)
-
Disruption of a GATA1-binding motif upstream of XG/PBDX abolishes Xga expression and resolves the Xg blood group system
- 2018
-
Ingår i: Blood. - : American Society of Hematology. - 0006-4971 .- 1528-0020. ; 132:3, s. 334-338
-
Tidskriftsartikel (refereegranskat)abstract
- The Xga blood group is differentially expressed on erythrocytes from men and women. The underlying gene, PBDX, was identified in 1994, but the molecular background for Xga expression remains undefined. This gene, now designated XG, partly resides in pseudoautosomal region 1 and encodes a protein of unknown function from the X chromosome. By comparing calculated Xga allele frequencies in different populations with 2612 genetic variants in the XG region, rs311103 showed the strongest correlation to the expected distribution. The same single-nucleotide polymorphism (SNP) had the most significant impact on XG transcript levels in whole blood (P 5 2.0 3 10222). The minor allele, rs311103C, disrupts a GATA-binding motif 3.7 kb upstream of the transcription start point. This silences erythroid XG messenger RNA expression and causes the Xg(a2) phenotype, a finding corroborated by SNP genotyping in 158 blood donors. Binding of GATA1 to biotinylated oligonucleotide probes with rs311103G but not rs311103C was observed by electrophoretic mobility shift assay and proven by mass spectrometry. Finally, a luciferase reporter assay indicated this GATA motif to be active for rs311103G but not rs311103C in HEL cells. By using an integrated bioinformatic and molecular biological approach, we elucidated the underlying genetic basis for the last unresolved blood group system and made Xga genotyping possible.
|
|
| 11. |
|
|
| 12. |
- Nilsson, Helena Jernmark, et al.
(författare)
-
The transcriptional coregulator NAB2 is a target gene for the Wilms' tumor gene 1 protein (WT1) in leukemic cells
- 2017
-
Ingår i: Oncotarget. - : Impact Journals, LLC. - 1949-2553. ; 8:50, s. 87136-87150
-
Tidskriftsartikel (refereegranskat)abstract
- The Wilms' tumor gene 1 (WT1) is recurrently mutated in acute myeloid leukemia. Mutations and high expression of WT1 associate with a poor prognosis. In mice, WT1 cooperates with the RUNX1/RUNX1T1 (AML1/ETO) fusion gene in the induction of acute leukemia, further emphasizing a role for WT1 in leukemia development. Molecular mechanisms for WT1 are, however, incompletely understood. Here, we identify the transcriptional coregulator NAB2 as a target gene of WT1. Analysis of gene expression profiles of leukemic samples revealed a positive correlation between the expression of WT1 and NAB2, as well as a non-zero partial correlation. Overexpression of WT1 in hematopoietic cells resulted in increased NAB2 levels, while suppression of WT1 decreased NAB2 expression. WT1 bound and transactivated the proximal NAB2 promoter, as shown by ChIP and reporter experiments, respectively. ChIP experiments also revealed that WT1 can recruit NAB2 to the IRF8 promoter, thus modulating the transcriptional activity of WT1, as shown by reporter experiments. Our results implicate NAB2 as a previously unreported target gene of WT1 and that NAB2 acts as a transcriptional cofactor of WT1.
|
|
| 13. |
- Nylander, Anja, et al.
(författare)
-
SMIM1, carrier of the Vel blood group, is a tail-anchored transmembrane protein and readily forms homodimers in a cell-free system
- 2020
-
Ingår i: Bioscience Reports. - 0144-8463. ; 40:5
-
Tidskriftsartikel (refereegranskat)abstract
- Antibodies to the Vel blood group antigen can cause adverse hemolytic reactions unless Vel-negative blood units are transfused. Since the genetic background of Vel-negativity was discovered in 2013, DNA-based typing of the 17-bp deletion causing the phenotype has facilitated identification of Vel-negative blood donors. SMIM1, the gene underlying Vel, encodes a 78-amino acid erythroid transmembrane protein of unknown function. The transmembrane orientation of SMIM1 has been debated since experimental data supported both the N- and C-termini being extracellular. Likewise, computational predictions of its orientation were divided and potential alternatives such as monotopic or dual-topology have been discussed but not investigated. We used a cell-free system to explore the topology of SMIM1 when synthesized in the endoplasmic reticulum (ER). SMIM1 was tagged with an opsin-derived N-glycosylation reporter at either the N- or C-terminus and synthesized in vitro using rabbit reticulocyte lysate supplemented with canine pancreatic microsomes as a source of ER membrane. SMIM1 topology was then determined by assessing the N-glycosylation of its N- or C-terminal tags. Complementary experiments were carried out by expressing the same SMIM1 variants in HEK293T/17 cells and establishing their membrane orientation by immunoblotting and flow cytometry. Our data consistently indicate that SMIM1 has its short C-terminus located extracellularly and that it most likely belongs to the tail-anchored class of membrane proteins with the bulk of the polypeptide located in the cytoplasm. Having established its membrane orientation in an independent model system, future work can now focus on functional aspects of SMIM1 as a potential regulator of erythropoiesis.
|
|
| 14. |
- Pettersson, Louise, et al.
(författare)
-
Comparison of RNA- and DNA-based methods for measurable residual disease analysis in NPM1-mutated acute myeloid leukemia
- 2021
-
Ingår i: International Journal of Laboratory Hematology. - : Wiley. - 1751-5521 .- 1751-553X. ; 43:4, s. 664-674
-
Tidskriftsartikel (refereegranskat)abstract
- Introduction: Reverse transcriptase quantitative PCR (RT-qPCR) is considered the method of choice for measurable residual disease (MRD) assessment in NPM1-mutated acute myeloid leukemia (AML). MRD can also be determined with DNA-based methods offering certain advantages. We here compared the DNA-based methods quantitative PCR (qPCR), droplet digital PCR (ddPCR), and targeted deep sequencing (deep seq) with RT-qPCR. Methods: Of 110 follow-up samples from 30 patients with NPM1-mutated AML were analyzed by qPCR, ddPCR, deep seq, and RT-qPCR. To select DNA MRD cutoffs for bone marrow, we performed receiver operating characteristic analyses for each DNA method using prognostically relevant RT-qPCR cutoffs. Results: The DNA-based methods showed strong intermethod correlation, but were less sensitive than RT-qPCR. A bone marrow cutoff at 0.1% leukemic DNA for qPCR or 0.05% variant allele frequency for ddPCR and deep seq offered optimal sensitivity and specificity with respect to 3 log(10) reduction of NPM1 transcripts and/or 2% mutant NPM1/ABL. With these cutoffs, MRD results agreed in 95% (191/201) of the analyses. Although more sensitive, RT-qPCR failed to detect leukemic signals in 10% of samples with detectable leukemic DNA. Conclusion: DNA-based MRD techniques may complement RT-qPCR for assessment of residual leukemia. DNA-based methods offer high positive and negative predictive values with respect to residual leukemic NPM1 transcripts at levels of importance for response to treatment. However, moving to DNA-based MRD methods will miss a proportion of patients with residual leukemic RNA, but on the other hand some MRD samples with detectable leukemic DNA can be devoid of measurable leukemic RNA.
|
|
| 15. |
|
|
| 16. |
- Svensson, Emelie, et al.
(författare)
-
Deregulation of the Wilms' tumour gene 1 protein (WT1) by BCR/ABL1 mediates resistance to imatinib in human leukaemia cells
- 2007
-
Ingår i: Leukemia. - : Springer Science and Business Media LLC. - 1476-5551 .- 0887-6924. ; 21:12, s. 2485-2494
-
Tidskriftsartikel (refereegranskat)abstract
- The Wilms' tumour gene 1 (WT1) protein is highly expressed in most leukaemias. Co-expression of WT1 and the fusion protein AML1-ETO in mice rapidly induces acute myeloid leukaemia (AML). Mechanisms behind expression of WT1, as well as consequences thereof, are still unclear. Here, we report that the fusion protein BCR/ABL1 increases expression of WT1 mRNA and protein via the phosphatidylinositol-3 kinase (PI3K)-Akt pathway. Inhibition of BCR/ABL1 or PI3K activity strongly suppressed transcription from WT1 promoter/enhancer reporters. Forced expression of BCR/ABL1 in normal human progenitor CD34+ cells increased WT1 mRNA and protein, further supporting the notion of BCR/ABL1-driven expression of WT1 in human haematopoietic cells. Forced expression of WT1 in K562 cells provided protection against cytotoxic effects of the ABL1 tyrosine kinase inhibitor imatinib, as judged by effects on viability measured by trypan blue exclusion, metabolic activity, annexin V and DAPI (4', 6-diamidino-2-phenylindole) staining. None of the isoforms provided any detectable protection against apoptosis induced by arsenic trioxide and only very weak protection against etoposide, indicating that WT1 interferes with specific apoptotic signalling pathways. Our data demonstrate that WT1 expression is induced by oncogenic signalling from BCR/ABL1 and that WT1 contributes to resistance against apoptosis induced by imatinib.
|
|
| 17. |
- Svensson, Emelie, et al.
(författare)
-
The Wilms' tumor gene 1 (WT1) induces expression of the N-myc downstream regulated gene 2 (NDRG2)
- 2007
-
Ingår i: DNA and Cell Biology. - : Mary Ann Liebert Inc. - 1044-5498 .- 1557-7430. ; 26:8, s. 589-597
-
Tidskriftsartikel (refereegranskat)abstract
- The Wilms' tumor gene 1 (WT1) protein is a transcriptional regulator that is highly expressed in immature hematopoietic progenitor cells and in the majority of patients with acute and chronic myeloid leukemia. However, it is still unclear how WT1 exerts its function(s) in hematopoietic cells. The aim of this work was to investigate the function of WT1 as a transcription factor in human hematopoietic progenitor cells. To this end, an oligonucleotide array approach was used to study the gene expression in CD34(+) cells from human cord blood retrovirally transduced with WT1 or a control vector. We found that the expression of the putative tumor suppressor gene N-myc downstream regulated gene 2 (NDRG2) mRNA was induced by WT1 in CD34(+) cells and also in leukemic U937 cells. Furthermore, a novel transcription start site in the NDRG2 gene was identified in WT1-transduced cells, in addition to two previously reported transcription start sites. These results show that the expression of the NDRG2 gene is directly or indirectly induced by WT1, and provide the first insights into transcriptional regulation of the NDRG2 gene, including demonstration of a novel splice variant.
|
|
| 18. |
- Ullmark, Tove, et al.
(författare)
-
Anti-apoptotic quinolinate phosphoribosyltransferase (QPRT) is a target gene of Wilms' tumor gene 1 (WT1) protein in leukemic cells
- 2017
-
Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 0006-291X. ; 482:4, s. 802-807
-
Tidskriftsartikel (refereegranskat)abstract
- Wilms' tumor gene 1 (WT1) is a zinc finger transcription factor that has been implicated as an oncogene in leukemia and several other malignancies. When investigating possible gene expression network partners of . WT1 in a large acute myeloid leukemia (AML) patient cohort, one of the genes with the highest correlation to . WT1 was quinolinate phosphoribosyltransferase (QPRT), a key enzyme in the . de novo nicotinamide adenine dinucleotide (NAD+) synthesis pathway. To investigate the possible relationship between . WT1 and . QPRT, we overexpressed . WT1 in hematopoietic progenitor cells and cell lines, resulting in an increase of . QPRT expression. WT1 knock-down gave a corresponding decrease in . QPRT gene and protein expression. Chromatin-immunoprecipitation revealed WT1 binding to a conserved site in the first intron of the . QPRT gene. Upon overexpression in leukemic K562 cells, QPRT conferred partial resistance to the anti-leukemic drug imatinib, indicating possible anti-apoptotic functions, consistent with previous reports on glioma cells. Interestingly, the rescue effect of QPRT overexpression was not correlated to increased NAD + levels, suggesting NAD + independent mechanisms. We conclude that . QPRT, encoding a protein with anti-apoptotic properties, is a novel and direct target gene of WT1 in leukemic cells.
|
|
| 19. |
- Ullmark, Tove, et al.
(författare)
-
Distinct global binding patterns of the Wilms' tumor gene 1 (WT1) -KTS and +KTS isoforms in leukemic cells
- 2017
-
Ingår i: Haematologica. - : Ferrata Storti Foundation (Haematologica). - 1592-8721 .- 0390-6078. ; 102:2, s. 336-345
-
Tidskriftsartikel (refereegranskat)abstract
- The zinc finger transcription factor Wilms' tumor gene 1 (WT1) acts as an oncogene in acute myeloid leukemia. A naturally occurring alternative splice event between zinc fingers three and four, removing or retaining three amino acids (+/-KTS), is believed to change the DNA binding affinity of WT1, although there are conflicting data regarding the binding affinity and motifs of the different isoforms. Increased expression of WT1 -KTS at the expense of WT1 +KTS isoform associates with poor prognosis in acute myeloid leukemia. We determined the genome-wide binding pattern of WT1 -KTS and WT1 +KTS in leukemic K562 cells by chromatin immunoprecipitation and deep sequencing (ChIP-seq). Motif discovery revealed distinct binding motifs for the isoforms, some of which have been previously reported as WT1 binding sites. We discovered that the WT1 -KTS isoform predominantly binds close to transcription start sites and to enhancers, in a similar fashion to other transcription factors, whereas WT1 +KTS binding is rather enriched within gene bodies. We observed a significant overlap between WT1 -KTS and WT1 +KTS target genes, despite the binding sites being distinct. Motif discovery revealed distinct binding motifs for the isoforms, some of which have been previously reported as WT1 binding sites. Additional analyses showed that both WT1 -KTS and WT1 +KTS target genes are more likely to be transcribed than non-targets, and are involved in cell proliferation, cell death, and development. Our study provides evidence that WT1 -KTS and WT1 +KTS share target genes yet still bind distinct locations, indicating isoform-specific regulation in transcription of genes related to cell proliferation and differentiation, consistent with involvement of WT1 in acute myeloid leukemia.
|
|
| 20. |
- Ullmark, Tove, et al.
(författare)
-
Global binding pattern of the Wilms' tumor gene 1 (WT1) +17AA -KTS isoform in leukemic cells
- 2016
-
Ingår i: Cancer Research. - 1538-7445. ; 76:14 Suppl.
-
Konferensbidrag (refereegranskat)abstract
- The aim of this study was to investigate the global DNA-binding pattern of Wilms' tumor gene 1 (WT1) in leukemic cells. Clinical and preclinical data indicate the zinc finger transcription factor WT1 as an oncogene, but the full target gene repertoire of WT1 in leukemic cells has not been previously characterized. The -KTS isoforms (excluding the three amino acid (KTS) insert between zinc finger three and four) are considered as the most efficient DNA-binders. Among these, the 17AA isoform (including 17 amino acids encoded by exon 5) is the most abundant one. To specifically analyze the DNA-binding of WT1(+17AA/-KTS) in leukemic cells, we generated a K562 clone that stably expressed BIO-tagged WT1(+17AA/-KTS), as well as the biotinylating enzyme Bir A. From the cells chromatin immunoprecipitation (ChIP) by streptavidin capture was performed followed by sequencing with a minimum of 50 million reads per sample. After alignment to the genome and peak calling, peaks were characterized and compared to available K562 tracks in the ENCODE database. We found that 45% of identified WT1(+17AA/-KTS) peaks are in the proximity of transcription start sites (promoter area, first exon or first intron) of target genes, whereas only 11% of randomized peaks were found here. Within the peaks we show strong enrichment for three different previously published WT1-binding motifs. Comparison to ENCODE tracks showed that WT1(+17AA/-KTS) peaks are in close proximity to binding sites of other transcription factors, to histone marks for actively transcribed genes, and to binding sites of chromatin modifiers. Considering peaks within promoters and gene bodies only (for safe assignment to a target gene), Gene Ontology (GO) analysis revealed enrichment of GO groups important for proliferation, cell death, embryonic development, and cell motility. In conclusion, WT1(+17AA/-KTS) binds close to transcription start sites in areas of active transcription. The target genes implicated in proliferation, cell death, cell signaling and motility adds to the growing evidence of WT1 as an effector gene in leukemia.
|
|
| 21. |
- Vidovic, Karina, et al.
(författare)
-
Erythroid Culture Based on Differentiation of Progenitor Cells from Leftover Buffy Coats Produced in the Reveos Automated Blood Processing System
- 2017
-
Ingår i: Vox Sanguinis. - : Wiley. - 1423-0410 .- 0042-9007. ; 112:supp 1, s. 288-288
-
Konferensbidrag (refereegranskat)abstract
- Background: Efforts to culture red blood cells (RBCs) for transfusion purposes are ongoing in numerous research laboratories worldwide. In addition, smaller-scale erythroid culture is one of many approaches toward better understanding of erythropoiesis, enucleation and RBC biology. Our center recently changed from traditional blood component separation to the Reveos(R) automated blood- processing system. Most buffy coats (BCs) after whole- blood donations were previously used for platelet production but leftover BCs are now discarded. BCs are still popular among scientists, mainly as a source of peripheral leukocytes but we asked the question if these leftover bags could be useful for erythroid culture. Aims: We aimed to set up and evaluate a protocol for in vitro erythroid culture of CD34+ progenitor cells extracted from Reveos(R) BCs. Methods: Total CD34+ cells in Reveos(R) BCs were measured using flow cytometry and the BD Cell Enumeration Kit. Following pre-enrichment and fractionation on a density gradient, the mononuclear cells were labeled with anti-CD34 and separated by magnetic particles using an EasySep™ magnet. Culture conditions for the extracted CD34+ cells were divided into phases I-III, each lasting for 7 days. Phase I is an expansion phase, phase II an erythroid differentiation phase and phase III a terminal differentiation phase. StemSpan™ SFEM II culture medium containing bovine serum albumin, human insulin and iron-saturated human transferrin was used in all phases. In phase I, SFEM II was supplemented with SCF, IL-3, FLT3L, TPO and dexamethasone, in phase II with SCF, IL-3, EPO and dexamethasone and in phase III with 30% serum, EPO and holo-transferrin. Fetal bovine serum (FBS) or human serum (HS) were used initially in parallel cultures, to compare their influence on proliferation rate and terminal differentiation. In all cultures, the total number of cells and cell viability were determined by cell counting in a Bürker chamber after trypan blue dye staining. The cultures were carefully monitored and diluted to 0.4 × 106/ml when entering phase II. Erythroid differentiation was analyzed on day 19, 21 and 23 by flow cytometry with anti-GPA-APC, anti-Band3-PE and anti- CD49d-PE-Cy7. The frequency of erythroblasts and enucleated cells present in the cultures were counted on May-Grünwald-Giemsa stained cytospin samples. Results: We found the CD34+ cell content by flow cytometry in BCs (n = 11, volume of 9.5-13.5 ml) to be 0.2-1.5 × 106 with a mean of 93% viability. Thereafter progenitor cells were isolated and subjected to a 3-phase erythroid culture procedure to evaluate their potential to expand and differentiate. Cells cultured in 30% HS in phase III were less viable but showed slightly higher co-expression of CD49d and Band 3 compared to cultures grown in 30% FBS (86% vs 75% on day 19, and 95% vs 86% on day 21), as well as a higher proportion Band 3-positive, CD49d-negative cells, indicating an increased proportion of late erythroblasts and reticulocytes. In order to promote end-stage maturation, HS cultures were favored. After 23 days in culture with HS in phase III, 8-21% of the cells were enucleated and the cultures contained ∼60% erythroblasts. Summary/Conclusions: Extraction of CD34+ hematopoietic progenitors at acceptable viability from leftover Reveos(R) BCs is feasible and convenient. Despite their small volume and the processing cycle, these BCs provide suitable starting material for small-scale, experimental erythroid cultures directly from single donors. Conditions in this culture model can be fine-tuned further to focus on different aspects of erythropoiesis.
|
|
| 22. |
- Vidovic, Karina, et al.
(författare)
-
Leukemia associated mutant Wilms' tumor gene 1 protein promotes expansion of human hematopoietic progenitor cells.
- 2013
-
Ingår i: Leukemia Research: A Forum for Studies on Leukemia and Normal Hemopoiesis. - : Elsevier BV. - 1873-5835 .- 0145-2126. ; 37:10, s. 1341-1349
-
Tidskriftsartikel (refereegranskat)abstract
- The transcription factor Wilms' tumor gene 1 (WT1) is highly expressed in the majority of leukemias, suggesting a role in leukemogenesis. Acquired WT1 mutations are reported as an independent predictor of poor clinical outcome, and mutations resulting in deletion of the entire DNA-binding zinc-finger domain (WT1delZ), is the most common type. The aim of this study was to study cellular effects of WT1(delZ) that may contribute to an oncogenic phenotype. We found that expression of WT1(delZ) supported proliferation of human hematopoietic CD34(+) progenitor cells. Moreover, WT1(delZ) transduced cells expressed erythroid markers, including raised levels of STAT5, independently of addition of erythropoietin. At the global gene expression level, WT1(delZ) caused upregulation of genes related to cell division and genes associated with erythroid maturation, in the absence of added erythropoietin. Our results indicate that WT1(delZ) promotes cell proliferation and expansion of progenitor cells, consistent with a possible role in leukemogenesis.
|
|
| 23. |
- Vidovic, Karina, et al.
(författare)
-
Wilms' tumor gene 1 protein represses the expression of the tumor suppressor interferon regulatory factor 8 in human hematopoietic progenitors and in leukemic cells.
- 2010
-
Ingår i: Leukemia. - : Springer Science and Business Media LLC. - 1476-5551 .- 0887-6924. ; 24:5, s. 992-1000
-
Tidskriftsartikel (refereegranskat)abstract
- Wilms' tumor gene 1 (WT1) is a transcription factor involved in developmental processes. In adult hematopoiesis, only a small portion of early progenitor cells express WT1, whereas most leukemias show persistently high levels, suggesting an oncogenic role. We have previously characterized oncogenic BCR/ABL1 tyrosine kinase signaling pathways for increased WT1 expression. In this study, we show that overexpression of BCR/ABL1 in CD34+ progenitor cells leads to reduced expression of interferon regulatory factor 8 (IRF8), in addition to increased WT1 expression. Interestingly, IRF8 is known as a tumor suppressor in some leukemias and we investigated whether WT1 might repress IRF8 expression. When analyzed in four leukemia mRNA expression data sets, WT1 and IRF8 were anticorrelated. Upon overexpression in CD34+ progenitors, as well as in U937 cells, WT1 strongly downregulated IRF8 expression. All four major WT1 splice variants induced repression, but not the zinc-finger-deleted WT1 mutant, indicating dependence on DNA binding. A reporter construct with the IRF8 promoter was repressed by WT1, dependent on a putative WT1-response element. Binding of WT1 to the IRF8 promoter was demonstrated by chromatin immunoprecipitation. Our results identify IRF8 as a direct target gene for WT1 and provide a possible mechanism for oncogenic effects of WT1 in leukemia.Leukemia advance online publication, 18 March 2010; doi:10.1038/leu.2010.33.
|
|
| 24. |
- Vidovic, Karina
(författare)
-
WILMS’ TUMOUR GENE 1 PROTEIN (WT1) – AN EFFECTOR IN LEUKEMOGENESIS?
- 2010
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Wilms’ tumour gene 1 (WT1) encodes a zinc-finger transcription factor functioning as a key regulator in organ development. WT1 was first identified as a tumour suppressor gene due to its inactivation in Wilms’ tumour cases, a childhood kidney cancer. In adult tissues WT1 expression is restricted to few organs, but various forms of cancers express high WT1 levels, suggesting an oncogenic potential for WT1. In normal hematopoiesis WT1 is expressed in a small subset of early progenitor cells, with declining levels as maturation occurs into mature blood cells. However, most primary leukemias express high levels of WT1 at diagnosis and this is an independent predictor of adverse outcome. In paper I, I demonstrate that WT1 expression is induced by tyrosine kinase signalling from BCR/ABL1 via the PI3K/Akt pathway. Chronic myeloid leukemia (CML) cells with forced expression of WT1 showed enhanced resistance to apoptosis induced by the ABL1 tyrosine kinase inhibitor, imatinib, further proposing an oncogenic function for WT1. Interferon regulatory factor 8 (IRF8), is absent or expressed at very low levels in leukemia. In paper II, IRF8 was shown to be a direct target gene of WT1, repressed by WT1 both in human hematopoietic progenitor cells and in leukemic cell lines. Furthermore, a strong anti-correlation between WT1 mRNA and IRF8 mRNA levels was observed when analyzed in a compiled compendium of expression datasets from primary leukemia samples. WT1 was retrovirally transduced in CD34+ progenitor cells to investigate the function of high WT1 levels in these cells. An oligonucleotide array revealed N-myc downstream regulated gene 2 (NDRG2), as an upregulated target gene to WT1. NDRG2 has later been identified as one of the genes in the HSC signature, which may suggest an indirect impact on HSC self-renewal capacity mediated by WT1 via regulation of NDRG2. In paper IV a delayed maturation of CD34+ human progenitor cells with forced expression of a WT1-mutant, lacking the entire zinc-finger domain and termed WT1(delZ), was observed. Enhanced and prolonged proliferation, increased clonogenic growth and differentiation towards an erythroid phenotype of WT1(delZ) expressing cells, suggest an oncogenic gain-of-function for mutated WT1, as compared to wild-type WT1. WT1 mutations are found in AML and T-ALL and are predominantly heterozygous frameshift mutations affecting exon 7, resulting in premature stop codons, thus encoding truncated WT1, similar to the WT1(delZ) protein. AML patients with WT1 mutations have higher frequency of resistence to chemotherapy with worse disease-free and overall survival rate. In summary, the abovementioned experimental findings, thoroughly discussed in my thesis, in association with the prevalence of high wild-type WT1 levels and WT1-mutations found in leukemias indicate an oncogenic effector role for WT1 in leukemogenesis.
|
|
| 25. |
- Westman, Julia S., et al.
(författare)
-
Allele-selective RUNX1 binding regulates P1 blood group status by transcriptional control of A4GALT
- 2018
-
Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 131:14, s. 1611-1616
-
Tidskriftsartikel (refereegranskat)abstract
- P1 and Pk are glycosphingolipid antigens synthesized by the A4GALT-encoded α1,4-galactosyltransferase, using paragloboside and lactosylceramide as acceptor substrates, respectively. In addition to the compatibility aspects of these histo-blood group molecules, both constitute receptors for multiple microbes and toxins. Presence or absence of P1 antigen on erythrocytes determines the common P1 (P1+Pk+) and P2 (P1-Pk+weak) phenotypes. A4GALT transcript levels are higher in P1 individuals and SNPs in non-coding regions of A4GALT, particularly rs5751348, correlate with P1/P2 status. Despite these recent findings, the molecular mechanism underlying these phenotypes remains elusive. The In(Lu) phenotype is caused by KLF1 haploinsufficiency and shows decreased P1 levels on erythrocytes. We therefore hypothesized KLF1 to regulate A4GALT expression. Intriguingly, P1 -specific sequences including rs5751348 revealed potential binding sites for several hematopoietic transcription factors, including KLF1. However, KLF1 binding did not explain P1-specific EMSA shifts and siRNA silencing of KLF1 did not affect A4GALT transcript levels. Instead, protein pull-down experiments using P1 but not P2 oligonucleotide probes identified RUNX1 by mass spectrometry. Furthermore, RUNX1 binds P1 alleles selectively and knockdown of RUNX1 significantly decreased A4GALT transcription. These data indicate that RUNX1 regulates A4GALT and thereby the expression of clinically important glycosphingolipids implicated in blood-group incompatibility and host-pathogen interactions.
|
|
