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| 2. |
- Järås, Marcus, et al.
(författare)
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Human short-term repopulating cells have enhanced telomerase reverse transcriptase expression.
- 2006
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Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 108:3, s. 1084-1091
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Tidskriftsartikel (refereegranskat)abstract
- Telomerase activity has been suggested to be critically involved in hematopoietic stem cell (HSC) self-renewal. However, it has been unclear whether human HSCs have telomerase activity and how telomerase activity is regulated within the HSC and progenitor pool. Here, we isolated living cord-blood (CB) CD34+ cells with up-regulated human telomerase reverse transcriptase (hTERT) expression by using an hTERT-reporting adenoviral vector encoding destabilized green fluorescent protein (dGFP) driven by the hTERT promoter, and functionally characterized them in comparison with control vector–transduced CD34+ cells expressing GFP. Following a 2-day serum-free transduction protocol, cells were sorted into a dGFP+ and a GFP+ fraction. Cell-cycle analysis revealed that the dGFP+ cells had a greater proportion of cells in S/G2/M phase compared with the GFP+ cells, (56% ± 1.8% vs 35% ± 4.3%; P < .001) and fewer cells in G0 phase (8.1% ± 3.0% vs 20% ± 4.7%; P < .01) However, the colony-forming and short-term nonobese diabetic/severe combined immunodeficient (NOD/SCID) B2m–/– mice bone marrow–repopulating capacities were similar between the dGFP+ and the GFP+ cells. Interestingly, the dGFP+ cells had a 6-fold lower repopulating capacity in NOD/SCID mice compared with the GFP+ cells and lacked secondary NOD/SCID B2m–/– mice bone marrow–repopulating capacity. Thus, up-regulation of hTERT expression within the CB HSC pool is accompanied by decreased self-renewal capacity.
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| 3. |
- Karlsson, Christine, et al.
(författare)
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Genetic intratumour heterogeneity in high-grade brain tumours is associated with telomere-dependent mitotic instability.
- 2007
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Ingår i: Neuropathology & Applied Neurobiology. - : Wiley. - 1365-2990 .- 0305-1846. ; 33:4, s. 440-454
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Tidskriftsartikel (refereegranskat)abstract
- Glioblastoma multiforme (GBM) and other high-grade brain tumours are typically characterized by complex chromosome abnormalities and extensive intratumour cytogenetic heterogeneity. The mechanisms behind this diversity have been little explored. In this study, we analysed the pattern of chromosome segregation at mitosis in 20 brain tumours. We found an abnormal segregation of chromatids at mitosis through anaphase bridging (10-25% of anaphase cells) in all 10 GBMs. Anaphase bridging was also found in two medulloblastomas (7-15%), one anaplastic astrocytoma (17%) and one oligodendroglioma (6%). These tumours showed a relatively high degree of cytogenetic complexity and heterogeneity. In contrast, cell division abnormalities were not found in low-grade brain tumours with less complex karyotypes, including two pilocytic astrocytomas and two ependymomas. Further analysis of two GBMs by fluorescence in situ hybridization with telomeric repeat probes revealed excessive shortening of TTAGGG repeats, indicating dysfunctional protection of chromosome ends. In xenografts established from these GBMs, there was a gradual reduction in cytogenetic heterogeneity through successive passages as the proportion of abnormally short telomeres was reduced and the frequency of anaphase bridges decreased from >25% to 0. However, bridging could be reintroduced in late-passage xenograft cells by pharmacological induction of telomere shortening, using a small-molecule telomerase inhibitor. Telomere-dependent abnormal segregation of chromosomes at mitosis is thus a common phenomenon in high-grade brain tumours and may be one important factor behind cytogenetic intratumour diversity in GBM.
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| 4. |
- Rebetz, Johan
(författare)
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Challenges in the treatment of malignant glioma with oncolytic adenovirus
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Malignant glioma is a tumor that spreads rapidly throughout brain and is almost impossible to remove completely by surgery. It is resistant to chemo- and radiotherapy and the prognosis is very poor. As a result, there is a desperate need for development of novel therapies against this devastating disease. Because of the heterogeneity in this cancer success has been limited, but a recent concept in cancer biology suggests that a small sub-population of cells within the tumor is responsible for initiation and maintenance of the whole tumor, the so-called cancer stem cells (CSC), a fact that could lead to new ways to treat cancer in the future. More specifically, a successful therapy has to target these CSC and in this way inhibit the re-growth of the tumor creating the possibilities for a more effective treatment against cancers such as malignant glioma. However, there is a lot of controversy surrounding this concept and in this thesis I have tried to clarify some of the uncertainties. Thus, since the CSC share many properties with normal neural stem cells (NSC) it could be that such a NCS gone awry is responsible for the CSC and the tumor. As a result, in my thesis, I have tried to look at cell surface markers for any similarities, or variations between the normal NSC and the CSC because this may give us an indication of the type of cells that are prone to transformation. In that case it would be easier to investigate which kind of changes lead to the development of a cancer cell. Additionally, in order to develop a new therapy against malignant glioma based on these possible findings we have chosen to use an adenovirus as a tool. Adenoviral infections are normally not dangerous to healthy individuals, and they are easy to manipulate so this makes them a good choice for cancer therapy. However, there is a common problem in viral treatment of cancer, which is one of the main reasons why treatments fail to give satisfactory results. This problem is the poor spread of the virus when injected into a tumor and I have as part of my work tried to identify mechanisms in order to solve it. Identifying such a mechanism can also answer some questions concerning the general problem in adenoviral biology, namely one of the reasons behind chronic adenoviral infections. We have discovered that during an adenovirus infection viral fiber proteins are secreted from the infected cell, which bind to and masks adjacent cells. Subsequently, because of this masking, as infected cells lyses and newly formed viral particles are released into the extra cellular space they infect new cells at a lower frequency. Identifying the type of cells where transformation takes place, and solving the problem of the poor spread of the virus when injected into a tumor, we hope to contribute to the development of new treatment regimes for malignant glioma in the future.
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| 5. |
- Rebetz, Johan, et al.
(författare)
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Fiber mediated receptor masking in non-infected bystander cells restricts adenovirus cell killing effect but promotes adenovirus host co-existence.
- 2009
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 4:12
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Tidskriftsartikel (refereegranskat)abstract
- The basic concept of conditionally replicating adenoviruses (CRAD) as oncolytic agents is that progenies generated from each round of infection will disperse, infect and kill new cancer cells. However, CRAD has only inhibited, but not eradicated tumor growth in xenograft tumor therapy, and CRAD therapy has had only marginal clinical benefit to cancer patients. Here, we found that CRAD propagation and cancer cell survival co-existed for long periods of time when infection was initiated at low multiplicity of infection (MOI), and cancer cell killing was inefficient and slow compared to the assumed cell killing effect upon infection at high MOI. Excessive production of fiber molecules from initial CRAD infection of only 1 to 2% cancer cells and their release prior to the viral particle itself caused a tropism-specific receptor masking in both infected and non-infected bystander cells. Consequently, the non-infected bystander cells were inefficiently bound and infected by CRAD progenies. Further, fiber overproduction with concomitant restriction of adenovirus spread was observed in xenograft cancer therapy models. Besides the CAR-binding Ad4, Ad5, and Ad37, infection with CD46-binding Ad35 and Ad11 also caused receptor masking. Fiber overproduction and its resulting receptor masking thus play a key role in limiting CRAD functionality, but potentially promote adenovirus and host cell co-existence. These findings also give important clues for understanding mechanisms underlying the natural infection course of various adenoviruses.
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| 6. |
- Rebetz, Johan, et al.
(författare)
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Glial Progenitor-Like Phenotype in Low-Grade Glioma and Enhanced CD133-Expression and Neuronal Lineage Differentiation Potential in High-Grade Glioma
- 2008
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:6, s. 1107-1107
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Tidskriftsartikel (refereegranskat)abstract
- Background: While neurosphere-as well as xenograft tumor-initiating cells have been identified in gliomas, the resemblance between glioma cells and neural stem/progenitor cells as well as the prognostic value of stem/progenitor cell marker expression in glioma are poorly clarified. Methodology/Principal Findings: Viable glioma cells were characterized for surface marker expression along the glial genesis hierarchy. Six low-grade and 17 high-grade glioma specimens were flow-cytometrically analyzed for markers characteristics of stem cells (CD133); glial progenitors (PDGFR alpha, A2B5, O4, and CD44); and late oligodendrocyte progenitors (O1). In parallel, the expression of glial fibrillary acidic protein (GFAP), synaptophysin and neuron-specific enolase (NSE) was immunohistochemically analyzed in fixed tissue specimens. Irrespective of the grade and morphological diagnosis of gliomas, glioma cells concomitantly expressed PDGFRa, A2B5, O4, CD44 and GFAP. In contrast, O1 was weakly expressed in all low-grade and the majority of high-grade glioma specimens analyzed. Co-expression of neuronal markers was observed in all high-grade, but not low-grade, glioma specimens analyzed. The rare CD133 expressing cells in low-grade glioma specimens typically co-expressed vessel endothelial marker CD31. In contrast, distinct CD133 expression profiles in up to 90% of CD45-negative glioma cells were observed in 12 of the 17 high-grade glioma specimens and the majority of these CD133 expressing cells were CD31 negative. The CD133 expression correlates inversely with length of patient survival. Surprisingly, cytogenetic analysis showed that gliomas contained normal and abnormal cell karyotypes with hitherto indistinguishable phenotype. Conclusions/Significance: This study constitutes an important step towards clarification of lineage commitment and differentiation blockage of glioma cells. Our data suggest that glioma cells may resemble expansion of glial lineage progenitor cells with compromised differentiation capacity downstream of A2B5 and O4 expression. The concurrent expression of neuronal markers demonstrates that high-grade glioma cells are endowed with multi-lineage differentiation potential in vivo. Importantly, enhanced CD133 expression marks a poor prognosis in gliomas.
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