In this study a CD31-expressing co-culture system was used to establish whether differential CD31/CD38 signalling may contribute to the poor prognosis associated with CD38 expression in CLL. Using western blot analysis, a PKB phospho-substrate antibody was used in combination with phospho-specific antibodies to identify ribosomal protein S6 and GSK3β as key signalling molecules that were augmented following short-term CD31-expressing co-culture. CD31-expressing co-culture did not alter the phosphorylation of STAT6. However, the addition of IL-4 to the cultures was a potent mediator of this signalling pathway. This highlights the specificity of signalling molecules to different external stimuli. Multi-colour flow cytometry was employed to quantify the expression of cell surface activation markers as well as intracellular phospho-proteins. The CD31-expressing co-culture led to a significant up regulation of the activation markers CD38, CD49d and CD69. Selective pharmacological inhibition of the phosphorylation of S6, STAT6 and ERK resulted in the down regulation of activation markers. Furthermore, the inhibition of p-STAT6 and p-ERK resulted in increased levels of apoptosis, which indicates that these signalling pathways are directly involved in CLL cell survival. Multi-colour flow cytometry was also used to quantitate the levels of phospho proteins, p-S6 and p-ERK. Similar to the results observed by antibody detection following western blotting, basal and inducible levels of p-S6 and p-ERK were elevated in primary CLL cells expressing high levels of CD38. Taken together, the work carried out in this project highlights the importance of using co-culture systems to stimulate CLL cells in vitro in order to mimic some of the key stimuli encountered in vivo. The dissection of the signalling pathways activated as a result of CD31/CD38 interactions provides a rational for the poor prognosis associated with elevated CD38 expression in this disease and identified candidate therapeutic targets that might particularly benefit this group of patients.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:600555 |
| Date | January 2013 |
| Creators | Betteridge, Sophie |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/58406/ |
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