The Cancer Stem Cell (CSC) hypothesis has provided a novel theory of tumorigenesis by suggesting that mechanisms of organogenesis in developmental processes may be aberrantly active in neoplasms. This hypothesis proposes that CSCS within a tumour play the role of stem cells in a tissue. This novel approach not only leads to new insights into the origination of cancer, but also suggests that CSCs may be responsible for the resistance of several cancer types to current therapies. Thus, CSCs may also be targets for novel therapies. This study interrogates the proposed role of Glioma Stem Cells (GSCs) in radioresistance of glioblastoma (GBM), and specifically addresses the cell cycle checkpoint responses of GSCs to ionising radiation (IR). The aims of this project are: to generate GSC cultures from primary and established GBM cell lines, to examine the radiosensitivity of GSCs, to investigate cell cycle and proliferation dynamics of GSCs and to investigate differential cell cycle checkpoint responses of GSCs to IR. In this study, a panel of glioblastoma cell lines was used, which included primary tumour cultures as well as established cell lines. Populations were enriched for GSCs by culturing as neurospheres in serum-free medium, or depleted of GSCs by culturing as adherent monolayers in serum- containing medium. Using flow cytometry, changes in the cell cycle progression of GSCs and non- stem glioma cells (NSGCs) after IR were compared. In contrast to previously published reports, GSCs did not show preferential activation of the G2-M checkpoint. However, in the 3 cell lines studied, GSCs exhibited earlier re-entry to mitosis than NSGCs. Results for G1-S arrest varied between the cell lines. To identify potential mechanisms for the early resumption of the cell cycle in GSCs, expression and phosphorylation of checkpoint proteins and the mitotic entry promoter Plk1 were investigated. The most likely explanation for the early G2/M checkpoint recovery in GSCs was reduced phosphorylation of Chk1. This hypothesis was validated by inhibiting Chk1, which led to an earlier release from IR-induced G2/M arrest in NSGCs, but did not change mitotic re-entry in GSCs. This study presents the first direct investigation of the effects of ionising radiation on cell cycle progression of CSCs. It also provides a detailed comparison of dynamic changes in radiation induced phosphorylation of checkpoint proteins in populations of GSCs and NSGCs. The novel observation that GSC show early release from the G2/M checkpoint is supported by reduced phosphorylation of Chk1 in these cells.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:618995 |
| Date | January 2011 |
| Creators | Yildirim, Salih |
| Publisher | University of Brighton |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://research.brighton.ac.uk/en/studentTheses/b2a074c1-d698-447c-b374-aa678a9cadf5 |
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