Cell-cycle checkpoint proteins help maintain genomic integrity by sensing damaged DNA and initiating DNA repair or apoptosis. Checkpoint protein activation to cell-cycle damaging agents can involve post-translational modifications and these alterations provide a means to determine whether DNA in a cell is damaged or not. Steinmoeller et al. (2009) showed that checkpoint proteins are suitable biomarkers for detecting genotoxins in Oncorhynchus mykiss (rainbow trout). In this project, two evolutionarily conserved checkpoint proteins, Rad1 and Hus1, have been cloned from rainbow trout and antibodies against these proteins were developed. This is the first time that either Rad1 or Hus1 has been characterized in rainbow trout. For rtRad1, it was determined that the open-reading frame was 840bp, which encodes 279aa with a predicted protein size of 31kDa. The rtRad1 amino-acid sequence is highly conserved and contains conserved exonuclease and leucine zipper domains. RT-PCR was used to identify alternatively spliced variants of rtRad1 and it appears that these variants encode different sized Rad1 proteins that are tissue and cell-line specific. A Rad1 splice variant that encodes an 18kDa protein appears to be abundant only in heart tissue and in the RTgill-W1 and RTbrain-W1 cell-lines. A genotoxicity study was completed where RTgill-W1 and RTbrain-W1 cells were treated with bleomycin, which induces double-stranded DNA breaks. In RTgill-W1, levels of an 18kDa Rad1 protein increased in a dose-dependent manner while in RTbrain-W1 the Rad1 levels remained the same. It appears that this 18kDa Rad1 protein may be directly involved in maintaining genomic integrity and shows potential to be used as a genotoxicity biomarker. This is the first time that an isoform of Rad1 has shown to be modified in the presence of a damaging agent. Both Rad1 and Hus1 need to be further characterized to determine their usefulness as genotoxicity biomarkers.

Cell-cycle checkpoint proteins help maintain genomic integrity by sensing damaged DNA and initiating DNA repair or apoptosis. Checkpoint protein activation to cell-cycle damaging agents can involve post-translational modifications and these alterations provide a means to determine whether DNA in a cell is damaged or not. Steinmoeller et al. (2009) showed that checkpoint proteins are suitable biomarkers for detecting genotoxins in Oncorhynchus mykiss (rainbow trout). In this project, two evolutionarily conserved checkpoint proteins, Rad1 and Hus1, have been cloned from rainbow trout and antibodies against these proteins were developed. This is the first time that either Rad1 or Hus1 has been characterized in rainbow trout. For rtRad1, it was determined that the open-reading frame was 840bp, which encodes 279aa with a predicted protein size of 31kDa. The rtRad1 amino-acid sequence is highly conserved and contains conserved exonuclease and leucine zipper domains. RT-PCR was used to identify alternatively spliced variants of rtRad1 and it appears that these variants encode different sized Rad1 proteins that are tissue and cell-line specific. A Rad1 splice variant that encodes an 18kDa protein appears to be abundant only in heart tissue and in the RTgill-W1 and RTbrain-W1 cell-lines. A genotoxicity study was completed where RTgill-W1 and RTbrain-W1 cells were treated with bleomycin, which induces double-stranded DNA breaks. In RTgill-W1, levels of an 18kDa Rad1 protein increased in a dose-dependent manner while in RTbrain-W1 the Rad1 levels remained the same. It appears that this 18kDa Rad1 protein may be directly involved in maintaining genomic integrity and shows potential to be used as a genotoxicity biomarker. This is the first time that an isoform of Rad1 has shown to be modified in the presence of a damaging agent. Both Rad1 and Hus1 need to be further characterized to determine their usefulness as genotoxicity biomarkers.

Progression through the cell cycle is tightly controlled, and the decision whether or not to enter a new cell cycle can be influenced by both internal and external cues. For budding yeast one such external cue is pheromone treatment, which can induce G1 arrest. Two distinct mechanisms are known to be involved in this arrest, one dependent on the arrest protein Far1 and one independent of Far1, but the exact mechanisms have remained enigmatic. The studies presented here further elucidate both of these mechanisms. We looked at two distinct aspects of the Far1-independent arrest mechanism. First, we studied the role of the G1/S regulatory system in G1 arrest. We found that deletion of the G1/S transcriptional repressors Whi5 and Stb1 compromises Far1-independent arrest, but only partially, and that this partial arrest failure correlates to partial de-repression of G1/S transcripts. Deletion of the CKI Sic1, however, is more strongly required for arrest in the absence of Far1, though not when Far1 is present. Together, this demonstrates that functionally overlapping regulatory circuits controlling the G1/S transition collectively provide robustness to the G1 arrest response. We also sought to reexamine the phenomenon of pheromone-induced loss of G1/S cyclin proteins, which we suspected could be another Far1-independent arrest mechanism. We confirmed that pheromone treatment has an effect on G1 cyclin protein levels independent of transcriptional control. Our findings suggest that this phenomenon is dependent on SCFGrr1but is at least partly independent of Cdc28 activity, the CDK phosphorylation sites in Cln2, and Far1. We were not, however, able to obtain evidence that pheromone increases the degradation rate of Cln1/2, which raises the possibility that pheromone reduces their synthesis rate instead. Finally, we also studied the function of Far1 during pheromone-induced G1 arrest. Although it has been assumed that Far1 acts as a G1/S cyclin specific CDK inhibitor, there has been no conclusive evidence that this is the case. Our data, however, suggests that at least part of Far1’s function may actually be to interfere with Cln-CDK/substrate interactions since we saw a significant decrease of co-pulldown of Cln2 and substrates after treatment with pheromone. All together, the results presented here demonstrate that there are numerous independent mechanisms in place to help robustly arrest cells in G1.

The coordination between cell growth and division is a highly regulated process that is intimately linked to the cell cycle. Efforts to identify an independent mechanism that measures cell size have been unsuccessful. Instead, we propose that size control is an intrinsic function of the basic cell cycle machinery. My work shows that in the fission yeast Schizosaccharomyces pombe Cdc25 accumulates in a size dependent manner. This accumulation of Cdc25 occurs over a large range of cell sizes. Additionally, experiments with short pulses of cycloheximide have shown that Cdc25 is an inherently unstable protein that quickly returns to a size dependent equilibrium in the cell suggesting that Cdc25 concentration is dependent on size and not time. Thus, Cdc25 can act as a sizer for the cell. However, cells are still viable when Cdc25 is constitutively expressed suggesting that there is another sizer in the case that Cdc25 expression is compromised. Cdc13 is a likely candidate due to the similar characteristics to Cdc25 and the ability to activate Cdc2. Cdc13 accumulates during the cell cycle in a manner similar to Cdc25. I show that in the absence of Cdc2 tyrosine phosphorylation, the cell size is sensitive to Cdc13 activity showing that Cdc13 accumulation can determine when cells enter mitosis. These results suggest a two sizer model where Cdc25 is the main sizer with Cdc13 acting as a backup sizer in the event of Cdc25 expression is compromised. Additionally, in the absence of Cdc2 phosphorylation by the kinases Wee1 and Mik1, mitotic entry is regulated by the activity of Cdc2. In the absence of Cdc2 phosphorylation, this activity is regulated by binding of cyclins to Cdc2. Under these circumstances, the activity of Cdc13 can regulate mitotic entry provide further evidence that Cdc13 could be a sizer of the cell in the case where Cdc25 expression is compromised. The results I present in this dissertation provide the groundwork for understanding how cells regulate size and how this size regulation affects cell cycle control in S. pombe . The results show how the intrinsic cell cycle machinery can act as a sizer for the G2/M transition in S. pombe . Interestingly, this mitotic commitment pathway is well conserved suggesting a general solution for size control in eukaryotes at the G2/M transition. Understanding the mechanism of how protein concentration is regulated in a size dependent manner will give much needed insight into how cells control size. Elucidating the mechanism for size control will capitalize on decades of research and deepen our understanding of basic cell biology.

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Yes / Topoisomerase inhibitors are in common use as chemotherapeutic agents although they can display reduced efficacy in chemotherapy-resistant tumours, which have inactivated DNA damage response (DDR) genes, such as ATM and TP53. Here, we characterise the cellular response to the dual-acting agent, Alchemix (ALX), which is a modified anthraquinone that functions as a topoisomerase inhibitor as well as an alkylating agent. We show that ALX induces a robust DDR at nano-molar concentrations and this is mediated primarily through ATR- and DNA-PK- but not ATM-dependent pathways, despite DNA double strand breaks being generated after prolonged exposure to the drug. Interestingly, exposure of epithelial tumour cell lines to ALX in vitro resulted in potent activation of the G2/M checkpoint, which after a prolonged arrest, was bypassed allowing cells to progress into mitosis where they ultimately died by mitotic catastrophe. We also observed effective killing of lymphoid tumour cell lines in vitro following exposure to ALX, although, in contrast, this tended to occur via activation of a p53-independent apoptotic pathway. Lastly, we validate the effectiveness of ALX as a chemotherapeutic agent in vivo by demonstrating its ability to cause a significant reduction in tumour cell growth, irrespective of TP53 status, using a mouse leukaemia xenograft model. Taken together, these data demonstrate that ALX, through its dual action as an alkylating agent and topoisomerase inhibitor, represents a novel anti-cancer agent that could be potentially used clinically to treat refractory or relapsed tumours, particularly those harbouring mutations in DDR genes.

Alzheimer's disease (AD) is the most common form of dementia. As the prevalence of AD increases with age, population aging will inevitably lead to an exponential increase in the proportion of older persons suffering from this disease. According to 2005 WHO estimate, 26.6 million people (approximately 0.55% of the general population) suffered from this disease. AD not only affects intellectual and functional abilities, it is also associated with significant neuropsychiatric disturbances. The pathogenesis of AD is characterized by widespread cerebral atrophy, abnormal deposition of amyloid plaques and tau protein in the central nervous system. While the classical histopathological features of AD are well recognized, exact physiological mechanisms that initiate the cascade of neural degeneration are still under active investigation. / As mentioned, the telomere length studies focused on ethically Chinese subjects recruited from two independent samples. The first clinical sample consisted of 411 older people and the other sample from healthy aging study, 976 community dwelling men were recruited. All subjects were assessed with the Cantonese version of the Mini-mental State Examination (CMMSE) for global cognitive function. Genomic DNA of the subjects was extracted from the peripheral whole blood sample. Lengths of the telomere were measured with Quantitative Real-Time PCR and the Ct ratio of the telomere and a control gene (36B4) of each sample was compared with the standard curve constructed with 4 selected sample's telomere lengths measured previously by Southern blotting. / For the first association study of the cell cycle checkpoint genes and AD, sample was recruited from a prospective study of cognitive function and risk factors for development of AD. 701 elderly were clinically evaluated for diagnosis of AD by psychiatrists. For this sample, genotyping of tagging SNPs of the 10 cell-cycle checkpoint genes were carried out by Restriction Fragment Length Polymorphism (RFLP) analysis. All tagging SNPs were selected from HapMap database and 5000bp upstream and downstream regions of each gene was also included. / For the results, the association study with cell cycle checkpoint genes, there was no SNPs found to be associated with diagnosis of clinical AD. We also found out that telomere length was associated with age in both two healthy aging men and clinical samples. There was no association between education and telomere lengths. For subjects in the healthy aging study, participants with CMMSE scores fell into the lowest 25% were found to have shorter telomere lengths. Similar result was found in the clinical AD sample. / In the study, telomere lengths were negatively associated with age. As the telomere will be shortened for each cell cycle, this finding correlated with physiological function at a cellular level. Statistical analysis also showed that shorter telomere lengths were found in subjects with poorer cognitive function. However, as age is a major determinant for cognitive impairments, further studies are recommended to evaluate the interaction effects of age in this association. Telomere shortening will cause cell senescence, and may be associated with faster neuronal degeneration, thus affecting cognitive function. Further studies should be conducted to examine its usefulness as an adjuvant biomarker for risk stratification of AD intervention trials. / Recent researches begin to unfold the physiological significance of telomere. A telomere is a repetitive region at the end of a chromosome. Basic functions of telomeres are involved with protection of the chromosome during replication and preventing chromosomal rearrangement or fusion. Abnormal telomere lengthening may be related to cancerous conditions. At a cellular level, telomere may also be related to aging and limitation in cell lifespan. In my study, I aimed to evaluate the association between the lengths of telomere and global cognitive function in community dwelling Chinese older persons in Hong Kong. As the length of telomere is also determined by the turnover rates of cells, apart from association study of telomere lengths and cognitive function, I also tried to study the association of genes related to cell cycles and AD. Polymorphisms of ten cell-cycle checkpoint genes, i.e. RB1, CDKN1A, CDK5R1, CDK2AP1, CDKN2A, CDKN2C, MDM2, P53, GSK3B, TPND1 and CDKN1B genes, were chosen in my project. / The thesis comprised of three studies. The first study was an association study of cell cycle checkpoint gene single nucleotide polymorphisms (SNPs) with clinical diagnosis of AD. The second study was an association study of telomere lengths and clinical diagnosis of AD in a clinical sample of patients suffering from the disease. The third study was an association study of the telomere lengths and global cognitive status in a group of active community dwelling older men who participated in a healthy aging study. / Lau, San Shing. / Adviser: Linda C.W. Lam. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 101-124). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.