Cremona, C.A.; (2009) The role of anchorage in cell cycle control. Doctoral thesis , UCL (University College London).

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Abstract

Mammalian cells generally require both mitogens and anchorage signals in order to proliferate. Failure to receive these signals results in either cell-cycle arrest or cell death, known as anoikis, due to activation of anchorage-dependent checkpoint mechanisms. Transformed cells alleviate these checkpoints, via activation of oncogenes and/or inactivation of tumour suppressors. In contrast, detachment of normal cells halts cell-cycle progression in G1, because of insufficient cyclin D1 induction to overcome the Rb/E2F checkpoint, accumulation of cyclin-dependent kinase inhibitors, and lack of cyclin A production. Because of the complexity of anchorage-dependent mechanisms involved, several changes are required for cells to become anchorage independent: impairment of both Rb and p53 pathways, plus activation of Ras. These specific defects are found in many human cancers, and anchorage independence correlates strongly with tumourigenic potential. Here I have used cells expressing SV40 LT, with and without oncogenic Ras, to model the changes leading to anchorage independent proliferation. Importantly, cells expressing SV40 LT alone retain their anchorage dependence, despite Rb and p53 inactivation. However, the mechanism responsible for this cell-cycle arrest in suspension is not known. Using immunoprecipitation and kinase assays, I demonstrated that the cyclin-dependent kinase inhibitor p27 curbs cell-cycle progression in these cells. FACS analysis showed that surprisingly, these cells do not undergo a robust checkpoint arrest, but instead stall throughout the cell cycle, showing abnormal DNA replication. Further investigation by metaphase spread showed the accumulation of aneuploid nuclei, indicating the development of genomic instability. This resulted in a higher rate of transformation among cells cultured without anchorage for a limited time. This work demonstrates that anchorage signals enable proper activation as well as assembly of cyclin-dependent kinase complexes, and that adhesion is particularly important for maintaining orderly cell-cycle progression and preventing genomic instability in checkpoint-deficient cells.

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