The role of intracellular signalling pathways in CHO cell growth in a synthetic environment

Industrial batch and fed-batch culture of Chinese hamster ovary (CHO) cell lines for the production of recombinant proteins, utilises the ability of engineered CHO cells to grow and proliferate in suspension in a synthetic environment that is free of many of the extracellular signals (e.g. exogenous growth factors, cell to matrix and cell to cell contacts) that control growth in vivo. This functional capability derives from extensive cell culture experimentation and lengthy adaptation processes to generate production cell lines. Many different approaches have been used in this study to develop an understanding of the underlying signalling pathway changes that facilitate CHO cell adaptation to suspension growth in a synthetic environment. Initial characterisation experiments compared the growth of a parental adherent CHO cell line (CCL-61) and a directly adapted suspension CHO cell line (CHO-SA) in different growth environments. Results from this characterisation showed that in this model system, unadapted CCL-61 cells are unable to grow and proliferate in suspension conditions, while suspension CHO-SA cells are able to grow and proliferate in suspension conditions but only when an exogenous growth factor, in this case Insulin, is present in the culture media. Differences in the PI3K/Akt and MAPK signalling pathway activation status between the unadapted CCL-61 and suspension adapted CHO-SA cell lines growing in adherent and suspension conditions were comparatively mapped by western blotting analysis and immunoprecipitation/immunoblot analysis. Results indicate that a shift in signalling flux occurs when cells are taken from an adherent growth environment to a suspension growth environment. In adherent cells, integrin-mediated attachment to the extracellular matrix stimulates an up-regulation in signalling flux via the MAPK pathway. When integrin-mediated attachment is abolished, as in suspension growth, a shift in signalling flux is seen towards the PI3K/Akt pathway. Further experimental data using chemical inhibitors against specific signalling intermediates such as PI3K, Akt and MEK 1/2 suggests that activation of these signalling intermediates is vital for cell survival and proliferation in differing synthetic environments and therefore they V are viable targets for genetic engineering strategies used to functionally substitute for extracellular signals. Confocal microscopy and flow cytometry were used to deduce how the PI3K/Akt and MAPK signalling pathways interact with specific cell surface membrane transducers such as integrins and specific receptor tyrosine kinases (RTKs) such as the insulin receptor. Analysis of integrin expression levels between adherent and suspension cell lines shows that despite the lack of integrin-mediated adhesion utilized by suspension cells, the expression levels of various integrins, inclucing β1, 5 and 1, are not down-regulated in suspension adapted CHO cells. Further analysis of the activation status of key adaptor proteins such as Focal Adhesion Kinase (FAK) demonstrates how changes at the cell surface are transmitted to downstream signaling networks.