Neuroblastoma (NB) is one the most common cancers in infancy and childhood, andpossession of amplified MYCN gene sequence (gene locus 2p24) is related toaggressive disease and poor prognosis, with a clear distinction established regardingthe survival of patients based on the gene copy-number of MYCN. However, theexpression of MYCN has been reported to vary between patients of even the sameNB subgroups and more importantly its significance in relation to NB prognosis isstill not clearly established with various reports presenting contradicting results.In this study, a bottom-up Systems Biology approach is suggested for studying boththe significance of MYCN expression, but also the distribution of control in therelated pathways that regulate this expression. An initial model was constructeddescribing the basic steps in MYCN expression and it was parameterised with valuesobtained from the literature. The results from the model simulations and analysisgenerated the hypothesis that the expression of MYCN cannot be used exclusively asa predictive factor without taking into account the relative levels of its dimerisationpartner, the protein MAX. Additionally, it was predicted that the amplification ofMYCN had a more pronounced relative effect at lower rather than at higher MYCNgene copy numbers.In order to create separate models for 4 NB cell-lines, it was necessary to performabsolute measurements for MYCN at the DNA, mRNA and protein level, as well asfor the MAX protein. The MYCN gene copy numbers were measured using theqPCR method, while a new data analysis method was suggested for performingabsolute quantification with the use of house-keeping genes, appropriate statisticalmethods and no reference samples. The relative amounts of MYCN mRNA werealso measured using qPCR and the results obtained were in agreement with thesuggested levels from the literature.The absolute measurement of the N-Myc and MAX proteins was attempted usingtwo complementary methods, western blots and ELISA. A series of optimisationexperiments and data analysis steps were taken that resulted in the refinement of theexperimental conditions to the point where they can be used for successfulquantification of the absolute levels of the N-Myc protein. Alternatively, theprocedure used for the MAX protein proved problematic and was not as successful.Overall, this study was successful in becoming the first step for an expanded bottomupsystems biology study regarding the significance of MYCN expression in NB.The combination of both the modelling and experimental parts of this workillustrated some of the potential benefits of Systems Biology approaches in studyingdisease. In this case the resulting model, once fully parameterised with experimentaldata, can be expanded in a number of suggested ways that address questions like therole and control of MYCN expression in relation to the cell-cycle deregulation ormulti-drug resistance in NB, giving in the process a better understanding regardingsuitable treatment targets for individual NB cases.
|Date of Award||31 Dec 2012|
- The University of Manchester
|Supervisor||Philip Day (Supervisor) & Hans Westerhoff (Supervisor)|