Function and regulation of human cyclin A

Cyclins regulate the progression through the cell cycle by binding and activating cyclin dependent kinases (CDK). Cyclin A is an essential gene that is expressed in cells from the beginning of S-phase until mitosis. Previously we have shown that cyclin A becomes unstable in prometaphase of mitosis and that it is degraded by a ubiquitin- and proteasome-dependent proteolysis. Furthermore we have found that the degradation of cyclin A in prometaphase is required for sister chromatid separation, the rapid degradation of cyclin B1 and exit from mitosis. Cyclin A is degraded as soon as the ubiquitin ligase APC/C gets activated while cyclin B1, another substrate of the APC/C, is protected from degradation until the metaphase to anaphase transition by the spindle assembly checkpoint. The molecular and mechanistic basis of the differences in the degradation of these two mitotic cyclins is not understood and its explanation might provide important insights into the mechanism of protein degradation. The biochemical function of cyclin A as an activator of CDKs is well established but the biological function of this kinase is not known. In addition, because tumour cells express high levels of cyclin A and interfering with its function has been shown to cause cell death cyclin A could be an important target for cancer therapy. To understand the mechanisms how proteins are recognised for targeted destruction we want to further explore cyclin A ubiquitination in mitosis (aim 1) and in G1 phase (aim2) by analysing the stability of cyclin mutants and cyclin chimeras in vitro and in vivo by using time lapse fluorescence video microscopy of GFP-tagged proteins. This analysis might also explain the apparent substrate specificity of the spindle assembly checkpoint. In addition we propose to generate human cyclin A `shut-off` cell lines to be able to define when during the cell cycle cyclin A performs its essential function (aim3).

Cyclin A is a conserved gene essential for cell cycle progression. We have set out to further study the expression, function and regulation of cyclin A in human cells. Elevated cyclin A expression has been reported for several different tumours and like other proliferation markers, such as Ki-67, high levels of cyclin A have been correlated with poor prognosis in patients. We have collaborated with others to analyse the expression of cyclin A in cases of Hodgkin lymphomas. Hodgkin lymphomas are mainly derived from B-cells, which form large multinucleated cells called Reed-Sternberg cells. These cells have a poor mitotic index and proliferate only slowly but produce cytokines that stimulate neighbouring normal lymphocytes to excessive proliferation. We In this studies unique Hodgkin tissue arrays, developed by Stephan Dirnhofer, Insitute of Pathology at the University of Basel, were used to stain Reed-Sternberg cells for proliferation markers, which revealed that 99 % of Hodgkin and Reed-Sternberg cells were strongly positive for cyclin A, while surrounding lymphocytes showed the expected cell-cycle restricted expression pattern of cyclin A. Thus, although these cells are not rapidly cycling, they are not quiescent as they express cyclin A, suggesting that these cells are arrested during the later stages of the cell cycle. How is cyclin A regulated in normal cells? Cyclin A expression is tightly regulated both at the transcriptional as well as posttranslational level. By using timelapse fluorescence videomicroscopy of living cells, we have found that constitutive expression of cyclin A - fused to green fluorescent proteins (GFP) to make is detectable in living cells, alone is not sufficient to prevent cyclin A oscillations, since cyclin A is degraded during early mitosis and reappears only briefly before the onset of S-phase. However, we have identified mutants of cyclin A that prevent its proteolytic degradation during G1-phase. In contrast to non-degradable cyclin A, expression of cyclin B1 was found to be essential for anaphase chromosome movements. We have identified the chromokinesin hKid as a downstream target of active Cdk1, which prevents anaphase chromosome movements. To be able to study the function of cyclin A, we have developed an efficient technology for conditional gene knockdown in human cells. This system consists of an inducible promoter to express a short RNA, which folds into a hairpin-like structure and is processed by an RNAse enzyme into small double-stranded RNAs known to induce RNA-dependent mRNA degradation. We could show that this inducible RNAi system is able to suppress the protein levels of cyclin A and inhibit proliferation of human tissue culture cells. Finally, because RNAi often fails to reveal a complete los-of- function phenotype we have developed several technologies - based on `recombineering`- for the rapid generation of gene-targeting vectors, which can be used to completely disrupt the function of genes, either in the mouse or in tissue culture cells. In summary, we have have developed several important technologies, such as stable conditional RNAi, that allow us to study the function of essential genes. In combination with conditional gene overexpresison and RNAi we could show that cyclin degradation is required for normal anaphase in human cells.