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fbtwitterlinkedinvimeoflicker grey 14rssslideshare1
Towner, Laurence
Languages: English
Types: Doctoral thesis
Subjects: QH426, R1
Activation of complement and its terminal pathway leads to the formation and insertion of the membrane attack complex (MAC) in the membranes of target cells. Complement is activated in tumours as is clear from the presence of complement activation products in cancer tissues. Over-expression of membrane bound complement regulators on tumour cells together with endogenous recovery mechanisms restricts complement activity and results in escape from lytic killing; nevertheless, sublytic MAC deposition is not without consequence. Sublytic MAC assembly on nucleated cells causes cell activation, secretion of extracellular matrix and pro-inflammatory cytokines and may cause protection from apoptosis. Signalling of these events is unclear.\ud The global effects of sublytic MAC were addressed in the murine colon carcinoma cell line (CT26) through the use of microarray technology. Cells were exposed to sublytic complement attack using pooled normal human serum (pNHS) and compared to MAC-inhibited controls generated using pNHS containing the C5 inhibitor OmCI. Total RNA was extracted at 0, 1 and 12 hours post-exposure and subjected to microarray analysis using the Illumina platform. Top statistically significant changes were then identified and a list of genes upregulated at both time points was uploaded to MetaCore and a gene network generated. From this a number of co-regulated genes which converged on the EGFR were highlighted. These were cxcl1, amphiregulin and matrix metalloproteinases (Mmp) 3 and 13. Both the top statistically significant and network derived genes were validated using qPCR. Changes in protein levels were then tested using western blot analyses for Mmp3 and Areg. Inhibition of the MEK/ERK, and to a lesser extent PI3K/Akt, signalling suppressed the gene upregulation that occurred in response to MAC but inhibition of p38 and JNK had no effect, implicating a MEK/ERK- PI3K co-activation. MAC deposition and not C5a/C5aR axis signalling was shown to be responsible for the mmp3 gene upregulation response.\ud Identification of MAC-mediated events and the signalling pathways involved may provide insight into the mechanisms by which complement activation influences tumour growth. In particular the data suggest that sublytic MAC deposition might promote a gene expression response which pushes the cells to a more aggressive phenotype by the upregulation of proliferative, survival, invasion and migratory signals. This in turn will inform strategies that seek to harness complement or complement regulation in tumour immunotherapy.

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