The evolutionary pressure to prevent re-replication by inactivating licensing during S phase leaves higher-eukaryotes with large genomes, such as human cells, vulnerable to replication stresses. Origins licensed in G1 must be sufficient to complete replication as new origins cannot be licensed in response to irreversible replication fork stalling. Interdisciplinary approaches between cellular biology and biophysics predict that replication of the genome is routinely incomplete in G2, even in the absence of external stressors. The frequency of converging replication forks that never terminate due to irreversible stalling (double fork stall), which result in a segment of unreplicated DNA, was modelled using high quality origin-mapping data in HeLa and IMR-90 cells. From this, hypotheses were generated that related an increase in unreplicated segments of DNA to reduced functional origin number. Presented in this thesis is the confirmation of this relation by quantifying chromosome mis-segregation and DNA damage responses when origin number was reduced using RNAi against licensing factors. The number of ultrafine anaphase bridges and 53BP1 nuclear bodies are in remarkable concordance with the theoretical predictions for the number of double fork stalls, indicating that cells are able to tolerate under-replication through such post-replicative cellular responses. 53BP1 preferentially binds to chromatin associated with large replicons, and functions synergistically with dormant origins to protect the stability of the genome. Additional candidates, inspired by common fragile site research, have also been characterised as responders to spontaneous under-replication, and include FANCD2 and MiDAS, which function in early mitosis to facilitate completion of replication before cells enter anaphase. In conclusion, a series of mechanisms that sequentially function throughout the cell cycle protects the stability of the human genome against inevitable spontaneous under-replication brought about by its large size.

Arsenic is a universal environmental toxicant associated mostly with skin related diseases in people exposed to low doses over a long term. Low dose arsenic trioxide (ATO) with long exposure will lead to chronic exposure. Experiments were performed to provide new knowledge on the incompletely understood mechanisms of action of chronic low dose inorganic arsenic in keratinocytes. Cytotoxicity patterns of ATO on long-term cultures of HaCaT cells on collagen IV was studied over a time course of 14 days. DNA damage was also assessed. The percentages of viable cells after exposure were measured on Day 2, Day 5, Day 8, and Day 14. Statistical and visual analytics approaches were used for data analysis. In the result, a biphasic toxicity response was observed at a 5 μg/ml dose with cell viability peaking on Day 8 in both chronic and acute exposures. Furthermore, a low dose of 1 μg/ml ATO enhanced HaCaT keratinocyte proliferation but also caused DNA damage. Global gene expression study using microarray technique demonstrated differential expressions of genes in HaCaT cell exposed to 0.5 μg/ml dose of ATO up to 22 passages. Four of the up-regulated and 1 down-regulated genes were selected and confirmed with qRT-PCR technique. These include; Aldo-Keto Reductase family 1, member C3 (AKR1C3), Insulin Growth Factor-Like family member 1 (IGFL1), Interleukin 1 Receptor, type 2 (IL1R2) and Tumour Necrosis Factor [ligand] Super-Family, member 18 (TNFSF18), and down-regulated Regulator of G-protein Signalling 2 (RGS2). The decline in growth inhibiting gene (RGS2) and increase in AKR1C3 may be the contributory path to chronic inflammation leading to metaplasia. This pathway is proposed to be a mechanism leading to carcinogenesis in skin keratinocytes. The observed over expression of IGFL1 may be a means of triggering carcinogenesis in HaCaT keratinocytes. In conclusion, it was established that at very low doses, arsenic is genotoxic and induces aberrations in gene expression though it may appear to enhance cell proliferation. The expression of two genes encoding membrane proteins IL1R2 and TNFSF18 may serve as possible biomarkers of skin keratinocytes intoxication due to arsenic exposure. This research provides insights into previously unknown gene markers that may explain the mechanisms of arsenic-induced dermal disorders including skin cancer / Environmental Sciences / D. Phil. (Environmental science)

Cellular senescence has been proposed to be equivalent to organismal aging and is one of the outcomes of the cell fate decision process in response to DNA damage that occurs in cells. When a cell encounters DNA damage, the cell cycle is immediately halted to evaluate which decision to take in response to genomic insult. The choices are between repairing the damage and continue division, or enter a non-replicative but viable state called senescence or to die if damage is severe (Figure 1). The signaling cascade, which detects this damage and regulates the cell fate decision, is collectively called as DNA damage response (DDR). However, the exact mechanism of how delineation for each decision happens is still not clear. Since DNA damage works as a mediator for cell fate decision, my work aimed to study senescence as a DNA damage response. In addition, the role of free radicals like ROS in cellular senescence is not very clear because though an increase in their concentrations is recorded in aged cells, it is not evident if the increase seen the cause or the effect of aging, primarily because they themselves capable of causing DNA damage. This conundrum have always led to confounding observations wrt role of free radicals in the cellular senescence process and if the senescence is caused through agents which rely on ROS to cause DNA damage, ROS becomes absolutely integral to the aging process. To understand this aspect formed the first line of investigation in my work along with identification of the sensor of DNA damage, which drives various cell fates. During organismal ageing there is an accumulation of senescent cells, which could be the major reason for functional decline of tissues and organs with age. However, to study changes associated with signaling molecules with respect to ageing, a cellular model system for senescence driven through DNA damage was needed, using which interplay between senescent / aged cells and cellular niche can be established. Studying the spatial and temporal alterations in signaling dynamics, within the cell as well as with the neighbouring niche during the senescence process in anticipated to provide us better understanding about the complex process of ageing. For this, the objectives were defined to establish and characterize the DNA damage induced senescence model using various parameters, and especially study the signaling dynamics of GPCR mediated signaling in senescence. The role of chemokine receptor, CXCR4 and its ligand, CXCL12 mediated signaling was chosen for the study. The following sections describe the findings that were obtained from the various objectives studied during the course of this study. Section 1. Development and characterization a model system to study cellular senescence as a DNA damage response. In this part of the study, I characterized genotoxic stress induced cellular senescence model using 5-Bromodeoxyrudine as the DNA damaging agent. BrdU, owing to its property of being a thymidine analogue, is incorporated in dividing cells, and this incorporation is recognized as DNA damage. This triggers ‘persistent’ DNA damage response signaling, including activation of ATM kinase, one of the primary DNA damage sensor. As anticipated, the DDR response detected was directly proportional to the dose of BrdU treatment and so was Reactive Oxygen Species (ROS) levels, a known senescence mediator. Using this model system of direct DNA damage mediated DDR activation and induction of cellular senescence, the growth-arrested cells were extensively characterized for presence and quantum of most of the senescence associated markers known in literature. BrdU treated cells, which became senescent showed presence of DNA damage, morphological changes like flat, enlarged, granule rich appearance, expression of senescence associated molecular markers like p21, IL8, showed senescence associated beta galactosidase activity, refractiveness to growth factor for division, increased ROS levels, Golgi dispersion, etc. The secretome of the treated cells also showed increased secretion of inflammatory cytokines which are attributed to a senescence phenotype, called as Senescence Associated Secretory Phenotype (SASP), which triggered proliferative and migratory effect on cancer cells. Overall, in this part of the study, it was established that BrdU can cause DNA damage and induce senescence as one of the cell fate in response to the intermediate dose of damage. The senescent cells generated in the model system was established to be akin to senescence observed by replicative exhaustion of normal cells, thereby making our model applicable to the physiological studies as well. Section 2. Insights into the role of ATM-ROS axis during senescence initiation and maintenance using DDR mediated cellular senescence model. While the BrdU model system for generating senescent cells was being developed and characterized, it was observed that there is an increase in ATM activation as well as ROS production concomitant to the a dose of BrdU. At the same time it was also observed that senescent cells showed persistent DDR signaling and high levels of ROS. Using this premise, in the second objective of my study I aimed to identify if ATM and ROS are critical during initiation of senescence, when the cells are insulted with the DNA damaging agent or during the maintenance of senescent state of the cells. By quenching ROS during the initiation state, I recorded that ROS is not critical for inducing senescence and perhaps the increase in ROS levels in senescent cells is due to their higher metabolic activity. By inhibiting ATM activation during DNA damage, it was observed that BrdU induces senescence through direct DNA damage, and active ATM and DDR signaling is absolutely critical for the senescence initiation. It was also established that ATM is not just a DNA damage sensor but also a redox regulator in the senescence model system. Prevention of ATM activation in presence of DNA damage blocked senescence initiation and also triggered increased ROS levels in the cells affecting their long term viability, suggesting ATM regulates ROS levels as well in addition to sensing DNA damage. In order to study the role of ATM-ROS axis in the maintenance of senescence state, already senescent cells were subjected to ROS quenching and/ or ATM inhibition and it was identified that both these signaling molecules are essential for maintaining the viability of senescent cells. The findings from these study thereby show that senescence can be divided into two temporally distinct stages, initiation or early senescence stage and second, maintenance stage of senescence. Overall, I was able to characterize the presence of temporally linked ROS – dependent and ROS – independent events in cellular senescence, which are independently mediated by ATM kinase (Figure 1). Dose of Genotoxic Stress damage DDR Senescence initiation Repair Cell cycle ATM arrest kinase Death Growth arrest Senescence maintenance Senescence Cell ROS viability Elevated metabolism Figure 1. Signaling cascades regulating senescence onset and maintenance mediated through DDR. Cells enter senescence state in response to DNA damage, depending on the dose of insult, through an ATM dependent and ROS independent pathway. Unlike this ATM-ROS axis is critical for the maintenance of senescent state of damaged but viable cells. Section 3. Understanding the role of CXCR4 – CXCL12 mediated signaling in senescence. Age dependent changes in cellular signaling are less explored and I was specifically interested in understanding how presence of senescent cells affects its microenvironment or vice versa i.e. how microenvironment affects senescent cells. In this premise the third objective of this study was defined towards identifying role of a GPCR, CXCR4 mediated signaling in cellular senescence and associated inflammation. CXCR4 is a ubiquitously expressed GPCR and it’s only known ligand is CXCL12/ SDF1 (stromal derived factor ), which is a homeostatic chemokine (i.e. its levels does not change under most physiological conditions). During characterization of DNA damage induced senescence model system, it was observed that this receptor expression is induced during DNA damage ells, which was also found to be so from data available from other gene expression studies as well. During the course of my work, I identified that senescent cells show CXCR4 up regulation in response to DNA damage, mediated through activation of ATM kinase - HIF1 axis and plays a critical role in enhancing the senescence associated inflammatory response in presence of its ligand, CXCL12. This CXCL12 dependent enhanced inflammatory response in damaged cells was determined to be sensitive to the pertussis toxin treatment and hence dependent on G protein activation. Further downstream analysis revealed the pro-inflammatory effect of the CXCR4 receptor activation was due to cAMP level suppression post activation by the Gi subunit. Given that cAMP levels are antagonistic to inflammatory phenotype, using a library of pharmacological compound library, I also discovered that cAMP specific PDE, phophodiesterase 4A, is also involved in regulating inflammatory response during the initiation stage of cellular senescence. The screen also confirmed the involvement of previously identified molecular components such as p38 MAPK and leukotrienes in the senescence associated inflammatory phenotype. The examination of the role of the CXCR4- CXCL12 axis in the deeply senescent cells surprisingly revealed that deeply senescent cells are refractory to CXCL12 stimulation in terms of inflammatory response, which was experimentally determined to be associated with impaired calcium release. Overall, the findings from this part of the study revealed a novel signaling cascade where CXCR4 up regulation is a part of the DDR response in cells, which utilizes the Local Excitation Global Inhibition (LEGI) mechanism to enhance the sensitivity of the damaged cells to its ligand CXCL12. This enhanced sensitivity mediates the CXCL12 dependent inflammatory response, which aids in attracting immune cells for clearance of these damaged cells. Once the cells have entered the senescent state, the axis is physiologically down modulated and the senescent cells showed refractiveness to CXCL12 stimulation, probably to prevent persistent acute inflammation, if the senescent cells are not cleared (Figure 2). Figure 2. CXCL12-CXCR4 axis in cellular senescence. During senescence initiation stage, when cells encounter DNA damage (Step 1), there is induction of CXCR4 receptor (Step 2), which enhances of CXCL12 mediated signaling for increased inflammatory response (Step 3). In the maintenance stage, where the cells are not cleared (Step 4), the axis is suppressed (Step 4), thereby bringing the levels of inflammatory secretome down, and thereby preventing damage to the cells (Step 5).

DNA damage in spermatozoa is a crucial contributor to spontaneous abortion, severe genetic disease in the offspring and infertility. The chromatin of spermatozoa is highly compacted, transcriptionally and translationally silent, hence lacking DNA damage response (DDR). DDR foci follow within seconds after a DNA double strand break (DSB) and correlate to an abortive topoisomerase-IIb activity during spermiogenesis. When comparing the DSB frequencies at the two most fragile genomic loci (fragile sites FRA3B, FRA16D) in human and murine spermatozoa with lymphocytes, significantly increased DSB levels were detected in spermatozoa in both species. This corroborates that spermatozoa are more prone to DSBs than somatic cells. When comparing the DSB frequencies at FRA3B/FRA16D in spermatozoa of smokers with non-smokers, two-fold increases were found, probably caused by cigarette smoke components triggering abortive topoisomerase-II¿ activity. The phosphorylated DDR proteins H2AX and ATM were identified in human spermatozoa and murine spermatids using multicolour immunostaining with laser-scanning confocal microscopy (LSCM) and Western blots. Based on significantly increased DDR foci in spermatozoa of smoking men, but lacking DDR foci in response to in vitro challenge with H2O2, an abortive topoisomerase-IIb activity is the likely cause of DDR foci in spermatozoa. As DDR foci are susceptible to cigarette smoke, they can potentially be used as a novel biomarker. When comparing paternal spermatozoa, and lymphocytes as well as maternal and cord lymphocytes from 39 families for DSBs (via high-throughput LSCM pH2AX detection) and DNA fragmentation (Comet assay), significant increases were found in newborns of mothers exposed to environmental tobacco smoke and smoking fathers. When challenging lymphocytes and spermatozoa to different genotoxicants, significantly increased DNA damage in newborns compared to adults was found. This confirms an exceptional vulnerability in newborns, believed to cause increased susceptibly to disease in later life, including cancer. / European Union¿s 6th Framework project Newborns and genotoxic exposure risk (NewGeneris), British Council¿s United Kingdom Indian Education Research Initiative (UKIER)

Thesis (PhD)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: The factors which control radiosensitivity are of vital importance for the understanding of cell inactivation and for cancer therapy. Cell cycle blocks, total induced DNA damage, DNA repair, apoptosis and chromatin structure are likely to playa role in the responses leading to cell death. I have examined aspects of irradiation-induced G2/M blocks in DNA damage and repair. In HT29, L132 and ATs4 cells the total amount of induced DNA damage by isodoses of 4.5 Gy, 5 Gy and 2 Gy was found to be 14 %, 14 % and 12 % respectively. Most of the DNA repair was completed before the G2/M maximum and only 3 % of DNA damage remains to be restored in the G2/M block. The radiosensitivity in eleven cell lines was found to range from SF2 of 0.02 to 0.61. By FADU assay the undamaged DNA at 5 Gy was found to range from 56% to 93%. The initial DNA damage and radiosensitivity were highly correlated (r2=0. 81). After 5 Gy irradiation and 12 hours repair two groups of cell lines emerged. The group 1 cell lines restored undamaged DNA to a level ranging from 94 % to 98 %. The group 2 cell lines restored the undamaged DNA to a level ranging from 77 % to 82 %. No correlation was seen between residual DNA damage remaining after 12 hours repair and radiosensitivity. In CHO-K1 cells chromatin condensation induced by Nocodazole was found to marginally increase the radiosensitivity as shown by the change of the mean inactivation dose (D) from 4.446 to 4.376 Gy. Nocodazole also increased the initial DNA damage, induced by 5 Gy, from 7 % to 13 %. In xrs1 cells these conditions increased the radiosensitivity from D of 1.209 to 0.7836 Gy and the initial DNA damage from 43 % to 57 %. Disruption of chromatin structure with a hypertonic medium was found to increase radiosensitivity in CHO-K1 cells from D of 4.446 to 3.092 Gy and the initial DNA damage from 7 % to 15 %. In xrs1 cells these conditions caused radiosensitivity to decrease from D of 1.209 to 1.609 Gy and the initial DNA damage from 43 % to 36 %. Repair inhibition by Wortmannin increased the radiosensitivity in CHO-K1 from a D of 5.914 Gy in DMSO controls to a D 3.043 Gy. In xrs1 cells repair inhibition had no effect on radiosensitivity. Significant inhibition of repair was seen in CHO-K1 at 2 hours (p<0.0001) and at 20 hours (p=0.0095). No inhibition of repair was seen in xrs1 cells at 2 hours (p=0.6082) or 20 hours (p=0.6069). While DNA repair must be allocated to the post-irradiation period, the G2/M block seen in p53 mutants reaches a maximum only 12 hours post-irradiation when most of the repair is completed. As the G2/M block resolves and cells reenter cycle 28 hours after the G2 maximum it appears that repair processes cannot be the only reason for the G2IM cell cycle arrest. At low doses of irradiation initial DNA damage correlates with radiosensitivity. This suggests that the initial DNA damage is a determinant for radiosensitivity. Repair of DNA double-strand breaks by the non-homologous end joining (NHEJ) mechanism, identified by inhibition with Wortmannin, was shown to influence residual DNA damage and cell survival. Both the initial DNA damage and DNA repair were found to be influenced by chromatin structure. Chromatin structure was modulated by high salt and by Nocodazole, and has heen identified as a parameter which influences radiosensitivity. / AFRIKAANSE OPSOMMING: Die faktore wat betrokke is in die meganisme van stralings-sensitisering is van hoogs belang vir die begrip van sel inaktiveering en kanker terapie. Sel siklus blokke, totale geïnduseerde DNS skade, DNS herstel, apoptose en chromatien struktuur is moontlike rol vertolkers in die sellulêre response wat ly tot seldood. Ek het die aspekte van stralings-geïnduseerde G2/M blokke in DNS skade en DNS herstelondersoek. Die hoeveelheid geïnduseerde DNS skade, deur ooreenstemmende stralings-dosisse, in HT29, L132 en ATs4 selle is 14 %, 14 % en 12 %. Meeste van die DNS herstel is klaar voordat die G2/M maksimum beryk word en net 3 % DNS skade blyoor om herstel te word in die G2/M blok. Die stralings-sensitiwiteit in elf sel lyne varieer tussen 'n SF2 van 0.02 en 0.61. Deur die gebruik van die FADU metode is gevind dat die onbeskadigde DNS na 5 Gy bestraling varieer tussen 56 % en 93 %. Die totale geïnduseerde DNS skade en stralings-sensitiwiteit was hoogs gekorreleer (r2=0.81). Na 5 Gy bestraling en 12 ure herstel kan die sel lyne in twee groepe gegroepeer word. Die groep 1 sellyne herstel die onbeskadigde DNS terug na 'n vlak wat varieer tussen 94 % en 98 %. Die groep 2 sel lyne herstel die onbeskadigde DNS terug tot op 'n vlak wat varieer tussen 77 % en 82 %. Geen korrelasie is gesien tussen oorblywende DNS skade en stralings-sensitiwiteit na 12 ure herstel nie. In die CHO-K1 sel lyn, chromatien kompaksie geïnduseer deur Nocodazole, vererger die stralings- sensitiwiteit soos gesien deur die gemiddelde inaktiveerings dosis (D) wat verlaag het van 4.446 tot 4.376. Nocodazole het ook die totale DNS skade verhoog van 7 % tot 13 %. Onder dieselfde kondisies, in die xrs1 sel lyn, is 'n verergering van stralings-sensitiwiteit (D) gesien van 1.209 tot 0.7836 en verhoog ONS skade van 43 % tot 57 %. Die ontwrigting van die chromatien struktuur deur die gebruik van hipertoniese medium het die stralings-sensitiwiteit (D) vererger in CHO-K1 selle van 4.446 tot 3.092. Die totale ONS skade is verhoog van 7 % tot 15 %. Onder dieselfde kondisies, in die xrs1 sellyn, verbeter die stralings-sensitiwiteit (D) van 1.209 tot 1.609 en die totale ONS skade verminder van 43 % tot 36 %. ONS herstel inaktiveering in die teenwoordigheid van Wortmannin het die stralings-sensitiwiteit (D) in CHO-K1 selle vererger van 5.914 in DMSO verwysings kondisies tot 3.043. Die ONS herstel inaktiveering in xrs1 selle het geen uitwerking gehaat op stralingssensitiwiteit nie. Noemenswaardige inaktiveering van ONS herstel is gesien in CHO-K1 selle na 2 ure (p<0.0001) en na 20 ure (p=0.0095). Geen inaktiveering is gesien in xrs1 selle na 2 ure (p=0.6082) of na 20 ure (p=0.6069) nie. TerwylONS herstel moet plaasvind na die bestralings periode, beryk die G2/M blok in p53 gemuteerde selle sy maksimum 12 ure na bestraling terwyl meeste van die ONS herstel alreeds voltooi is. Aangesien die G2/M blok eers 28 ure later begin sirkuleer moet die G2/M blok nog 'n funksie vervul anders as ONS herstel. By lae dosisse van bestraling korreleer die totale geïnduseerde ONS skade met stralings-sensitiwiteit. Dit dui daarop dat die totale ONS skade 'n bepalende faktor moet wees in stralings-sensitiwiteit. Die herstel van ONS skade deur die nie-homoloë eindpunt samevoeging (NHES) meganisme, geïdentifiseer deur inaktiveering deur Wortmann in, het 'n invloed op oorblywende ONS skade en sellulêre oorlewing. Beide die totale ONS skade en ONS herstel was beïnvloed deur die chromatien struktuur. Chromatien struktuur was gemoduleer deur hoë sout konsentrasies en deur Nocodazole, en is geïdentifiseer as a belangrike parameter wat stralings-sensitiwiteit beïnvloed.

In the context of the somatic mutation theory of chemical carcinogenesis, mutations are thought to arise during the replication of DNA past carcinogen-DNA adducts. The work described in this thesis deals with the testing of a hypothetical mechanism whereby mammalian cells are able to replicate their DNA past polycyclic aromatic hydrocarbon DNA adducts. The second objective of this thesis work was to develop both in vivo and in vitro models to study the induction of mutations in a target human gene by chemical carcinogens from two different classes, polycyclic aromatic hydrocarbons and nitrosamines. To approach the hypothetical mechanism of bypass replication in mammalian cells, synchronized Chinese hamster ovary cells were treated with the ultimate carcinogenic form of benzo (a) pyrene, 7β, 8α-dihydroxy-9α, 10α-epoxy-7,8,9,10-tetrahydrobenzo (a) pyrene (BPDE I). Using the pH step alkaline elution assay, it was found that the reduced rate of S phase progression was due to a delay in the appearance of multiple replicon size nascent DNA. It was determined using agarose gel electrophoresis that the ligation of Okazaki size replication intermediates was blocked in BPDE I-treated, synchronized CHO cells. The data obtained were, therefore, supportive of the 'block-gap' model of DNA bypass replication in carcinogen damaged mammalian cells. To study mutagenesis of a specific sequence induced by chemical carcinogens, the human c-Ha-ras proto-oncogene was transfected into the mouse fibroblast cell line, NIH 3T3. Transfected NIH 3T3 cell lines (HHRN 1-4) were isolated that had a low copy number of the human c-Ha-ras proto-oncogene and a non-transformed phenotype. It was determined that the integrated human c-Ha-ras gene was hypomethylated, and expressed at the messenger level. The human c-Ha-ras protein, p21, was also detected in these transfected cell lines. Treatment of the HHRN cell lines with the nitrosamine, N-methyl-N-nitroso-N'-nitroguanidine (MNNG) resulted in transformed NIH 3T3 foci. In vitro MNNG treatment of the plasmid, z-6, and transfection into NIH 3T3 cells led to the isolation of transformed cell lines. Screening of the in vitro and in vivo treated, transformed cell lines by RNA:RNA duplex mismatch analysis led to the detection of no mutations within the first exon of the human c-Ha-ras oncogene.

The type 1 insulin-like growth factor receptor (IGF-1R) is a receptor tyrosine kinase that mediates diverse cellular functions including growth, differentiation, migration and apoptosis protection. IGF-1R signalling has been implicated in tumorigenesis in a variety of cancers, and IGF-1R inhibitory drugs are currently undergoing clinical evaluation. Previous work in our laboratory has shown IGF-1R over-expression in urological cancers at both the mRNA and protein level, thus making it a potential therapeutic target. The first aim of this project was to develop a protocol for IGF-1R immunohistochemistry, investigate the expression and cellular distribution of the IGF-1R receptor in clear cell renal cell carcinomas (ccRCC), and assess correlation with clinical parameters. In tissue microarray analysis, IGF-1R was detected in ~90% of 195 ccRCCs, with signal in the plasma membrane, cytoplasm and also in the nucleus. The presence of nuclear IGF-1R in up to 50% of ccRCCs and its association with adverse prognosis was a novel finding, and suggests that nuclear IGF-1R may influence ccRCC biology. Further investigations will clarify its role in the nucleus and its potential as a prognostic biomarker. The second aim was to investigate effects of IGF-1R inhibition on radiosensitivity and DNA repair, following previous work in our laboratory showing that IGF-1R depletion enhances chemo- and radio-sensitivity, delays double strand break (DSB) resolution, and may play a role in the homologous recombination (HR) pathway of DNA DSB repair. However, the repair defect seen in these early experiments was larger than could be entirely explained by a defect in HR. The current project used a small molecule IGF-1R tyrosine kinase inhibitor AZ12253801 (AstraZeneca), which blocked IGF-1 induced IGF-1R activation and inhibited cell survival. AZ12253801 enhanced the radiosensitivity of prostate cancer cells, which appeared to be independent of effects of IGF-1R inhibition on cell cycle distribution and apoptosis induction. IGF-1R inhibition delayed the resolution of γH2AX foci, supporting a potential role for the IGF-1R in DSB repair. This delay in focus resolution was apparent at early time-points (less than 4 hr), and was epistatic with DNA dependent protein kinase (DNAPK) inhibition in prostate cancer cells and DNAPK deficiency in glioblastoma cells. These results suggest a role for the IGF-1R in the non-homologous end-joining (NHEJ) pathway of DNA DSB repair. A cell-based reporter assay in HEK-293 cells confirmed that IGF-1R inhibition suppressed DSB repair by NHEJ, helping to explain the radiosensitization demonstrated upon IGF-1R inhibition. There was lack of support for a transcriptional effect, with no significant change observed in gene expression on microarray analysis. Although the mechanism of this effect remains unclear, the observed inhibition of NHEJ has implications for the use of IGF-1R inhibitors in combination with DNA damaging agents in cancer treatment.

RASSF1A is a tumour suppressor, the silencing of which occurs through promoter methylation in a variety of human cancers. Loss of RASSF1A is associated with decreased sensitivity to DNA damaging agents and worse prognosis in breast, colon and lung cancers amongst others. RASSF1A functions in a number of cellular processes, promoting apoptosis in response to DNA damage or death receptor signalling, or cell cycle arrest at both G1/S and pro-metaphase checkpoints. As a scaffold protein, RASSF1A imparts these functions through direct interaction with target proteins. We have identified a novel interaction between RASSF1A and the SRC activator, OSSA. Further studies identify a role for RASSF1 in SRC signalling. We find that a second isoform of RASSF1, RASSF1C, the expression of which is maintained in cancers, is able to activate SRC. We also identify a novel tumour suppressor role for RASSF1A inhibiting SRC activation through binding of RASSF1C. SRC activation by RASSF1C expression promotes internalisation of adherens junctions leading to subsequent loss of tight junctions and cell polarity markers from sites of cell-cell contact. -catenin is also found to be re-localised throughout the cells from where it is hypothesised to be able to upregulate pro-proliferative genes. In addition, we find that RASSF1C expression promotes cell motility in both scratch wound and transwell assays. Finally, we show that RASSF1C expression enhances tumour cell aggressiveness using a mammosphere growth assay. We conclude that RASSF1C is an oncogene that can promote EMT through the activation of SRC family kinases. This function is inhibited by the tumour suppressor RASSF1A. This work highlights why RASSF1A is lost through epigenetic mechanisms and not mutation and why loss of RASSF1A is associated with more aggressive, metastatic cancers.

In the present study DNA damage was measured in peripheral blood lymphocytes from polyposis coli and colorectal cancer patients, treated with different dietary and environmental compounds and compared with lymphocytes from healthy individuals. In addition, confounding factors such as age, gender, alcohol intake and smoking habits were taken into consideration. The assays used in this study included the Comet assay, the Micronucleus assay, the Micronucleus-FISH assay and the sister chromatid exchange assay. The food mutagens, PhIP and IQ, as well as titanium dioxide nanoparticles (TiO2 NPs) induced a dose dependent increase in the DNA damage and chromosomal abnormalities in all tested groups regardless of confounding factors. Prior to experiments physicochemical characterisation of nanoparticles was conducted. In the presence of the flavonoids, quercetin and rutin that were acting in an antioxidant manner, the DNA damage resulting from the highest doses of food mutagens was significantly reduced. Thus, dietary supplementation with flavonoid-rich vegetables and fruits may prove very effective in protection against oxidative stress. The polyposis coli and colon cancer patients were more susceptible to food mutagens, PhIP and IQ, as well as TiO2 NPs, and in the majority of cases had a higher level of DNA damage in the Comet assay and higher cytogenetic damage in the Micronucleus assay. In the final project, twelve frequently encountered (NewGeneris) chemical compounds were evaluated to establish their damaging potential in lymphocytes and spermatozoa from healthy donors. The highest damage was produced by DNA reactive aldehydes, food mutagens and benzo[a]pyrene when assessed with the neutral and alkaline Comet assay with and without metabolic activation.

The synthesis and characterisation of novel bisnaphthalimidopropyl polyamine (BNIPP) derivatives, has gained pace over the last couple of years, as they have enhanced aqueous solubility, without loss of biological activity, in contrast to parent bisnaphthalimide derivatives. Recent work has shown that bisnaphthalimidopropyl spermidine (BNIPSpd) bis-intercalates to DNA, induces oxidative DNA damage, depletes polyamine levels and causes cell death, by apoptosis, in human colon cancer CaCO-2 and HT-29 cells. The aim of this thesis was to synthesise new BNIPP derivatives to highlight the important structural features required for biological activity, particularly, bisnaphthalimidopropyl functionality, and investigate their subsequent modes of action in breast cancer MDA-MB-231 and breast epithelial MCF-10A cells. Initially, work focused on determining the DNA binding affinities and biological activity of BNIPP derivatives. All BNIPP derivatives, except bisphthalimidopropyl diaminodecane (BPHPDadec) and mononaphthalimidopropylamine (NPA) (Δ Tm values of 15.8 and 10.2 °C, respectively, C50 values of > 10 μM, IC50 values of > 40 μM), exhibited strong DNA binding affinities and cytotoxic properties in both cell lines. Results indicate that BNIPP derivatives interact with DNA by bis-intercalation suggesting, therefore, that BNIPP derivatives target DNA. For the first time, an investigation into the mechanism of cellular entry, via the polyamine transport (PAT) system, was studied. However, none of the BNIPP derivatives utilised the MGBG-specific PAT system, suggesting that BNIPP derivatives utilise other modes of cellular entry. Two BNIPP derivatives, BNIPSpd and BNIPDaCHM, were further investigated, and results show that these derivatives significantly induced a dose dependent increase in DNA strand breaks from ≥ 0.1 μM, after 4 hours. BNIPSpd and BNIPDaCHM (at non toxic concentrations) also inhibited the repair of oxidative (H2O2) and methylative (MMS)-induced DNA strand breaks. Based on phosphatidylserine exposure and membrane integrity analyses, early apoptotic cell death was determined as a mode of cell death utilised by both BNIPSpd and BNIPDaCHM (5 μM), after only 0.5 hours treatment in MDA-MB-231 cells. Interestingly, BNIPDaCHM was identified, using HDAC assay kits, as a potent and selective SIRT2 enzyme inhibitor, thus, identifying, a novel structural backbone for the selective inhibition of HDAC enzymes.