ABSTRACTGlucocorticoids have long been used in the treatment of acute lymphoblastic leukaemia due to their ability to cause cell cycle arrest and apoptosis of lymphoid cells. However, some patients do not respond to glucocorticoid treatment and the majority, who initially respond, may relapse upon prolonged hormone treatment. The inefficiency of the treatment is mainly attributed to the gradual loss of the cellular sensitivity to glucocorticoid-induced apoptosis. Therefore, the need to understand the molecular mechanisms of resistance/ sensitivity of acute lymphoblastic leukaemia cells to glucocorticoid-induced apoptosis is of vital importance, as this will help to develop better prognostic outcomes and improve glucocorticoids therapy.Several mechanisms have been proposed to explain the evasion of glucocorticoid mediated apoptosis in resistant cells. These include post-translational modifications of GR especially phosphorylation which modulates the GR transcriptional activity, and GR mediated signalling thereby affecting gene expression and hence the balance between pro- and anti-apoptotic Bcl-2 family members. In addition the concentration of components of the energy metabolism pathways (i.e. oxidative phosphorylation and glycolysis) and ROS generation are altered in the acute lymphoblastic leukaemia cells. The hypothesis that differentially phosphorylated GR in the resistant versus sensitive ALL cells modulate GR transcriptional activity and target selectively resulting in diverse pro- or anti-apoptotic Bcl-2 family members' gene expression in the two cell lines was tested. Furthermore, in a similar manner, the possibility that differential GR phosphorylation diversely affected gene expression of GR transcriptional target genes that are components of cellular energy production pathways in resistant versus sensitive cells, altering energy and ROS production levels in distinct ways in the two cell lines was explored.GR was found to be predominantly phosphorylated at S211 in the glucocorticoid-sensitive CEM C7-14, and at S226 in the glucocorticoid-resistant CEM C1-15 cells. Differential GR phosphorylation is presumably an indication of dominant p38 MAPK activity in CEM C7-14 and JNK kinase activity in CEM C1-15, which could lead to adverse gene expression of some pro- and anti-apoptotic Bcl-2 family members and particularly Mcl-1, in the two cell lines. Furthermore, differential GR phosphorylation at S211 and S226 in CEM C7-14 and CEM C1-15 affected the gene expression of the Cytochrome C Oxidase assembly factors Surf-1 and SCO2 as well as the nuclear encoded Cytochrome C Oxidase subunit COX-Va and the mitochondrial encoded COX-I, COX-II and COX-III. This effect was more pronounced in the glucocorticoid-sensitive CEM C7-14 cells, probably due to the fact that GR was predominantly phosphorylated at S211 and hence transcriptionally active in these cells. Moreover, in comparison to the resistant CEM C1-15 cells, the CEM C7-14 cells exhibited higher levels of ROS, increased number of active mitochondria and up-regulated glycolysis upon inhibition of oxidative phosphorylation. Glucocorticoids further reduced ROS levels in the CEM C1-15 cells, and increased the NADH/ NAD+ ratio. In conclusion results presented in this thesis provide evidence that differential GR phosphorylation in resistant versus sensitive to glucocorticoid induced apoptosis cells plays essential role in the regulation of programmed cell death and energy metabolism pathways, offering a potential explanation for the molecular events that determine resistance / sensitivity to glucocorticoid-induced apoptosis in ALL cells.
|Date of Award||31 Dec 2012|
- The University of Manchester
|Supervisor||Costas Demonacos (Supervisor) & Marija Krstic-Demonacos (Supervisor)|
- glucocorticoids, resistance, sensitivity, apoptosis, acute lymphoblastic leukaemia
- ROS, energy production pathways, GR phosphorylation