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Poly(ADP-ribose) polymerase (PADPRP) is a nuclear enzyme with a well documented role in DNA repair. Inhibitors of PADPRP, (e.g. 3' substituted benzamides) potentiate the cytotoxicity of a wide range of antitumour drugs. The results presented in this thesis represent, to the best of my knowledge, the first comprehensive and quantitative assessment of the ability of a range of P ADPRP inhibitors to modulate the cellular responses to damaging agents. Two novel PADPRP inhibitors, 8-hydroxy-2-methyl quinazolin-4(3H)-one (NU1025) and 3,4 dihydro-5-methoxyisoquinolin-1-(2H)-one (PD 128763) were compared with two "classical" PADPRP inhibitors, 3-aminobenzamide (3AB) and benzamide (BZ). The relative potencies for 3AB, BZ, NU1025 and PD 128763 as PADPRP inhibitors in vitro were 1.0, ~1.0, ~43 and ~53 respectively. All compounds potentiated the growth inhibition and cytotoxicity of the monofunctional alkylating agent temozolomide (TM) in L1210 cells. For example, 10/-lM NUI025 and PD 128763 gave dose enhancement factors (DEF) of ~2 at 100/0 survival, whereas ImM 3AB and 0.5mM BZ where required to give similar DEF values. Cellular NADl- levels were depleted up to 50% by 1-2mM TM and this depletion was completely prevented by coincubation with 50-100µM PD 128763 and 1-3mM 3AB. TM induced DNA single strand break levels were increased in a concentration dependent manner by the P ADPRP inhibitors. Overall, the relative potencies for ability of the compounds to potentiate TM induced growth inhibition, cytotoxicity and DNA single strand breaks showed good correlation with those determined in an in vitro inhibition study, with both NU1025 and PD 128763 exhibiting ~60 fold increased inhibitory activity as compared to 3AB. The PADPRP inhibitors per se did not effect the growth or survival of the L 121 0 cells, nor increase DNA strand breakage. NAD+ is the substrate for PADPRP. A L1210 cell line made resistant to tiazofurin (TZ) utilising a step wise selection protocol was shown to be deficient in nicotinamide mononucleotide adenyl transferase (NMNAT) , the final enzyme required for NAD+ biosynthesis. The consequences of a reduced NMNAT activity (<3% of the parental line ) and an ~40% reduction in intracellular NAD+ levels were determined. The resistant cells showed an ~3 fold increased sensitivity to TM as compared to the parental cells. Upon coincubation with increasing concentrations of NU1025 in the presence of a fixed concentration of TM, growth inhibition was potentiated ~70 fold in the resistant cells but only ~10 fold in the parental cell line, demonstrating the reduced level of competition between NAD+ and NUI025 for PADPRP. However, DNA single strand breaks were increased in the resistant compared to the parental cell line only when NU1025 was coincubated with TM. In contrast, in the presence of the PADPRP inhibitors alone, equivalent growth inhibitory effects were observed in each of the cell lines, suggesting inhibition of PADPRP was not the cytotoxic effector. The ~40% NAD+ depletion observed could therefore suggest, that NAD+ levels in the resistant cells were reduced to, or near to the KmNAD+ for PADPRP.

Undergraduate honors paper--Mount Holyoke College, 2005. Dept. of Chemistry. / Includes bibliographical references (leaves 107-111).

Gliomas are common primary brain tumours that occur as low-grade (LGG) and high-grade gliomas (HGG). Typically occurring in younger adults, LGG has an indolent course with a median survival of 5-10 years, but carries an inherent potential for transforming into HGG. The thesis focused on LGG in adults, with the aim of identifying prognostic biomarkers for LGG. Paper I. Epileptic seizures are common symptoms in LGG. In a retrospective study, the correlation between 11C-methionine (MET) uptake, measured by Positron Emission Tomography (PET), and seizure activity was assessed in 101 patients with LGG. Although there was no correlation between MET uptake and seizure activity, survival was longer in patients who were seizure-free before surgery. Paper II. This finding prompted the search for common genetic pathways for both tumour and seizure development and a review of genetic polymorphisms in focal epilepsy and glioma risk. Cell cycle and immune response genes affecting both glioma and seizure risk were identified, and genes involved in synaptic transmission presented potential candidates for future studies. Paper III. The transcription factor PROX1 plays a pivotal role in normal development and carcinogenesis of various organs. The prognostic value of PROX1, together with established clinical and molecular prognostic factors for survival, was retrospectively assessed in 116 patients with LGG. High PROX1 expression in the tumour was associated with shorter survival. Paper IV. DNA repair enzymes, such as ERCC6, are crucial for maintaining genomic stability in glioma response to radiotherapy. An association between the polymorphism rs4253079, mapped to ERCC6, and longer survival in patients with LGG and HGG was identified. Paper V. As LGG typically presented as non-contrast enhancing tumours on morphological MRI (magnetic resonance imaging), the value of combined MET PET with physiological MRI for preoperative diagnosis was assessed in a prospective study of 32 patients with suspected LGG. Representative tumour areas were identified through a combination of perfusion-MRI with MET PET, which can be used as a baseline investigation for follow-up over time. Conclusions: The parameters seizure-freedom before surgery, the polymorphism rs4253079 in ERCC6 and low PROX1 expression in the tumor were identified as favorable prognostic biomarkers.

T cells have a crucial role in viral clearance and vaccine response; however, the mechanisms regulating their responses to viral infections or vaccinations remain elusive. In this study, we investigated T-cell homeostasis, apoptosis, DNA damage, and repair machineries in a large cohort of subjects with hepatitis C virus (HCV) infection. We found that naive CD4 T cells in chronically HCV-infected individuals (HCV T cells) were significantly reduced compared with age-matched healthy subjects. In addition, HCV T cells were prone to apoptosis and DNA damage, as evidenced by increased 8-oxoguanine expression and γH2AX/53BP1-formed DNA damage foci—hallmarks of DNA damage responses. Mechanistically, the activation of DNA repair enzyme ataxia telangiectasia mutated (ATM) was dampened in HCV T cells. ATM activation was also diminished in healthy T cells exposed to ATM inhibitor or to HCV (core protein) that inhibits the phosphoinositide 3 kinase pathway, mimicking the biological effects in HCV T cells. Importantly, ectopic expression of ATM was sufficient to repair the DNA damage, survival deficit, and cell dysfunctions in HCV T cells. Our results demonstrate that insufficient DNA repair enzyme ATM leads to increased DNA damage and renders HCV T cells prone to apoptotic death, which contribute to the loss of naive T cells in HCV infection. Our study reveals a novel mechanism for T-cell dysregulation and viral persistence, providing a new strategy to improve immunotherapy and vaccine responses against human viral diseases.