It is well established that cyclin and cyclin-dependent kinase (CDK) form complex that plays a central role in driving cell cycle progression. The fundamental functions of CDK and cyclin are well conserved across eukaryotes. However, gene families encoding the two type of proteins are significantly expanded in multicellular organisms compared with single-cell species. Despite intensive studies on CDK and its associated cyclin in cultured cell lines, especially in cancer cell lines, the partnership between individual CDKs and cyclins remains elusive especially in vivo. Here I present our preliminary results on establishing the molecular function of a well-conserved cyclin L encoded by cyl-1 in C. elegans. Human cyclin L was demonstrated to form a complex with both CDK11 and CDK12, but its association with the latter remains controversial. Despite a possible function in both transcription and pre-mRNA splicing as suggested by in vitro studies or in yeast, the in vivo function of cyclin L has yet been established in any species. To study cyl-1's function in vivo, we generated multiple strains each expressing a chromosomally integrated single-copy transgenes consisting of CYL-1::GFP flanked by its native regulatory sequences using miniMos technique. The transgene demonstrates ubiquitous expression in nuclei across developmental stages and cell types with few exceptions, including maturing oocytes, in which gene activity is known to be shut down, consistent with its function in transcription and splicing. Co-immunoprecipitation followed by mass spectrometry reveals that CYL-1 interacts with both CDK-11 and CDK-12 along with some other uncharacterized factors. Functional validation of these interactions is underway.

<p>Breast cancer is one of the most common malignancies in women. Due to early detection and the use of screening programs approximately 60% of all new cases lack lymph node involvement. Today, a substantial proportion of these women will be offered adjuvant systemic chemotherapy. However, better proliferation markers are needed to predict patient outcome and to avoid overtreatment. </p><p>Cyclin A, cyclin E and Ki-67 are all markers for proliferation and involved in the regulation of the cell cycle. Overexpression has been associated with disease recurrence in several studies, but the results have not been consistent. However, none of these studies has investigated aberrant expression of cyclin E (the expression of cyclin E during phases of the cell cycle other than late G1 and early S-phase). Studies have shown that aberrant cyclin E might provide additional prognostic information compared to cyclin E alone.</p><p>The aims of this thesis were 1.to investigate the prognostic value of cyclin A, cyclin E and aberrant cyclin E in early breast cancer. 2.to validate the tissue microarray (TMA) technique for cyclin A and 3.to define the most optimal cut-off values for cyclin A and Ki-67.</p><p>We found that the agreement of TMA and large section results was good with kappa values 0.62-0.75 and that the reproducibility of the two readers’ results was good or even very good, with kappa values 0.71 – 0.87. </p><p>The optimal cut-off value for cyclin A average was 8% and for cyclin A maximum value 11%. The corresponding values for Ki-67 were 15 and 22%. </p><p>Neither cyclin E nor aberrant cyclin E was a prognostic factor in low-risk node negative breast cancer patients. </p><p>Finally, we conclude that cyclin A is a prognostic factor in node negative breast cancer (univariate analysis average value OR=2.9 95% CI 1.8-4.6; maximum value OR=3.7 95% CI 2.3-5.9).</p>

Breast cancer is one of the most common malignancies in women. Due to early detection and the use of screening programs approximately 60% of all new cases lack lymph node involvement. Today, a substantial proportion of these women will be offered adjuvant systemic chemotherapy. However, better proliferation markers are needed to predict patient outcome and to avoid overtreatment. Cyclin A, cyclin E and Ki-67 are all markers for proliferation and involved in the regulation of the cell cycle. Overexpression has been associated with disease recurrence in several studies, but the results have not been consistent. However, none of these studies has investigated aberrant expression of cyclin E (the expression of cyclin E during phases of the cell cycle other than late G1 and early S-phase). Studies have shown that aberrant cyclin E might provide additional prognostic information compared to cyclin E alone. The aims of this thesis were 1.to investigate the prognostic value of cyclin A, cyclin E and aberrant cyclin E in early breast cancer. 2.to validate the tissue microarray (TMA) technique for cyclin A and 3.to define the most optimal cut-off values for cyclin A and Ki-67. We found that the agreement of TMA and large section results was good with kappa values 0.62-0.75 and that the reproducibility of the two readers’ results was good or even very good, with kappa values 0.71 – 0.87. The optimal cut-off value for cyclin A average was 8% and for cyclin A maximum value 11%. The corresponding values for Ki-67 were 15 and 22%. Neither cyclin E nor aberrant cyclin E was a prognostic factor in low-risk node negative breast cancer patients. Finally, we conclude that cyclin A is a prognostic factor in node negative breast cancer (univariate analysis average value OR=2.9 95% CI 1.8-4.6; maximum value OR=3.7 95% CI 2.3-5.9).

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A key effect of human papillomavirus (HPV) infection is to disrupt the normal cell cycle in order to subvert the cellular DNA replication machinery. Morphologically, condylomata induced by high and low risk HPV types cannot be distinguished and many studies have shown that the pattern of viral gene expression is similar in condylomata caused by both high risk and low risk HPV types. Detailed morphological study of cell cycle protein expression has not previously been performed on condylomata infected with low risk HPV types. The findings presented suggest that the mechanisms employed by low risk HPV6 or HPV11 to subvert cellular functions in condylomata acuminata are similar to those employed by high risk HPVs, with the exception of cyclin D1 and p53 protein over-expression. The differences in p53 expression and cyclin D1 expression seen between high and low risk HPV infection, reflect the known differences between high and low risk types and are in agreement with the known differences between high risk and low risk E6 and E7 proteins. PHK transduction studies demonstrated HPV E6 and E7 induce changes in cell cycle protein expression and that there are differences in cell cycle abrogation between HPV6 and HPV16. Disruption of the p53-MDM2 interaction can lead to activation of the p53 pathway. HPV infected lesions almost always contain wild-type p53. The binding of HPV E6 to p53, and its subsequent targeting for degradation, prevents activation of the p53 pathway in HPV infected cells. Cells over expressing HPV genes were treated with Nutlin-3, a MDM2-small molecule antagonist. The findings presented suggest treatment with Nutlin-3 induces cell cycle arrest in cells expressing HPV16 E7 and HPV6 E6 and HPV6 E7. This suggests a potential role for Nutlin-3 in the treatment of HPV infected cells.

Cyclin-Dependent Kinase 11 (CDK11) is a serine/threonine kinase encoded at human locus 1p36.3 by two paralogous genes CDK11A and CDK11B. CDK11 has diverse roles in the regulation of transcription, splicing, apoptosis and mitosis. In proliferating cells, two predominant isoforms are expressed: CDK11p58 and CDK11p110. CDK11p110 is expressed throughout the cell cycle and regulates transcription and splicing. CDK11p58 is expressed at mitosis via IRES-dependent translation; it mediates mitotic progression and faithful chromosome segregation. Loss of Cdk11 in murine models causes early embryonic lethality, demonstrating that CDK11 is essential for normal development. Furthermore, dysregulated CDK11 expression is associated with numerous late-onset disease states, indicating its importance in adult life. In cancer, abnormal expression of CDK11 correlates with poor prognosis in a variety of tumours. Moreover, deletion of the chromosomal region 1p36.3, containing the CDK11 locus, is frequently observed in cancer and has recently been identified in a case of the development disorder, Cornelia de Lange Syndrome (CdLS). This thesis aimed to examine the functions of CDK11 and the impact of their dysregulation in cancer and developmental phenotypes. The initial aim was to investigate the novel role for CDK11 in regulating autophagy in cancer cells; CDK11 depletion causes a marked autophagy phenotype, with accumulation of autophagy protein LC3. I demonstrate that this CDK11-mediated autophagy occurs as a consequence of mitotic dysregulation. Subsequently, I examined the role of autophagy following aberrant mitosis and chromosome missegregation. I show that autophagy is important in the maintenance of aneuploid karyotypes, with loss of autophagy impairing the survival of aneuploid cell populations. I then investigated the effects of CDK11 in regulating cancer cell motility and determined that CDK11 depletion retards cancer cell migration. However, I was unable to identify any failure in cell adhesion or cell polarization to explain this migration phenotype. Subsequently, I interrogated the CDK11 interactome to further characterize the mechanisms through which CDK11 regulates both novel and established functions. This work indicated the involvement of the distinct CDK11 isoforms in pathways that have not previously been reported. This included the interaction of CDK11p110 with ribosomal and spliceosomal proteins during mitosis and the interaction of CDK11p58 with spliceosomal and proteosomal constituents also during mitosis. These findings may provide the foundation for further study. Finally I describe work undertaken to sequence the CDK11 locus in a cohort of CdLS patients, with no known causative genetic mutation, to investigate CDK11A/CDK11B as candidate disease-associated genes. Although no causative mutation in CDK11A or CDK11B was identifying, sequencing of this region indicated NCBI and UCSC genome assemblies of this locus were inaccurate due to the genomic duplication. This has been confirmed by others and corrected in the most recent genome assemblies.

In the budding yeast Saccharomyces cerevisiae, the mitotic cell cycle is regulated by the cyclin-dependent kinase (CDK) Cdc28. Cdc28 is activated by binding to one of nine cyclins, which then directs Cdc28’s function and localization. Clb2 is the main mitotic cyclin, promoting entry into mitosis and progression from the metaphase to anaphase transition. In order for cells to exit mitosis, CDK activity must decrease; CDK activity is regulated through Clb2 degradation. Degradation of Clb2 is mediated by the Anaphase-Promoting Complex (APC), which is an E3 ubiquitin ligase that is regulated by the spindle assembly checkpoint. The APC has two activators: Cdc20 and Cdh1; APC-Cdc20 recognizes an N-terminal destruction box (D box) motif in Clb2 during anaphase, and APC-Cdh1 recognizes a KEN box motif at the onset of telophase. Activation of the spindle assembly checkpoint or inactivation of the APC causes cells to arrest in metaphase and, thus, results in accumulation of Clb2. While performing analysis of the function of Clb2 in regulation of mitosis, a lower molecular weight population of Clb2 was identified that seems to correspond to a cleaved form of Clb2 (p45Clb2). Both full-length Clb2 (p56Clb2) and truncated Clb2 (p45Clb2) have different accumulation patterns during either activation of the spindle assembly checkpoint or inactivation of the APC. Our current hypothesis is that p45Clb2 is cleaved between the D box and the KEN box and thus cannot be recognized by APC-Cdc20 but can still be ubiquitinated by APC-Cdh1. p45Clb2 also lacks a nuclear-export signal (NES), which causes its accumulation in the nucleus. We hypothesize that Clb2 cleavage constitutes a mechanism to ensure presence of high CDK activity to delay exit from mitosis.