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Exposure to the anticonvulsant valproic acid (VPA) is associated with a 7.5% rate of major malformations and a 1-2% incidence of neural tube defects (NTDs). Although the teratogenic outcomes resulting from VPA use during pregnancy were first identified in the 1980s, the mechanisms by which VPA induces birth defects are not fully elucidated. Based on evidence in the literature, the studies in this thesis examined the role of in utero VPA exposure on oxidative stress and epigenetic alterations in the developing embryo to provide further mechanistic insight into VPA’s teratogenic pathway. The first study investigated the role of oxidative stress in VPA-induced teratogenesis. Using CD-1 mice, catalase was shown to protect against VPA-induced effects on developmental and morphological parameters in both whole embryo culture and in vivo models. Studies in whole embryo culture demonstrated that markers of oxidative damage were not altered by VPA; however, VPA increased apoptosis in the neuroepithelium, which was attenuated by the addition of catalase. The second objective addressed epigenetic modifications induced by VPA in an in vivo mouse model. Maternal administration of VPA resulted in increased acetylation of histones H3 and H4, increased methylation of histone H3K4, and decreased methylation of histone H3K9. Furthermore, these changes were localized to VPA target tissues including the neuroepithelium, heart, and somites. Global DNA methylation in the embryo was not altered by VPA. The final objective was to determine VPA’s effect on a marker of DNA damage, markers of cell cycle proteins, and a marker of apotosis in vivo. Maternal administration of VPA resulted in a rapid increase of γH2A.X, a marker of DNA double strand breaks (DSBs). Increased expression of p27KIP1, a cyclin-dependent kinase inhibitor, and activated caspase-3, a marker of apoptosis, were observed and these changes were localized to the neuroepithelium of developing embryos. In conclusion, this thesis supports the hypothesis that VPA-induced increases in ROS production and HDAC inhibition may lead to altered gene expression patterns and consequently teratogenic effects, namely NTDs. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2011-05-24 13:12:33.778

Title of Dissertation: FUNCTION OF INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN 7(IGFBP7) IN HEPATOCELLULAR CARCINOMA By Dong Chen. Purpose: Hepatocellular carcinoma (HCC) is a highly virulent malignancy with no effective treatment, thus requiring the development of innovative and effective targeted therapies. The oncogene Astrocyte Elevated Gene-1 (AEG-1) plays a seminal role in hepatocarcinogenesis and profoundly downregulates Insulin-like Growth Factor Binding Protein-7 (IGFBP7). The present study focuses on analyzing potential tumor suppressor functions of IGFBP7 in HCC and the relevance of IGFBP7 downregulation in mediating AEG-1 function. Experimental Design: IGFBP7 expression was detected by immunohistochemistry in HCC tissue microarrays by real-time PCR and ELISA in human HCC cell lines. Dual Fluorescence in situ hybridization was performed to detect loss of heterozygosity at the IGFBP7 locus. Stable IGFBP7- overexpressing clones were established in the background of AEG-1- overexpressing human HCC cells and were analyzed for in vitro proliferation, senescence, in vivo tumorigenesis and angiogenesis. HCC cell lines infected with an adenovirus expressing IGFBP7 (Ad.IGFBP7) were analyzed by using in vitro cell cycle, apoptosis, in vivo tumorigenesis assays. Results: IGFBP7 expression is significantly downregulated in both human HCC patients’ samples and cell lines compared to normal liver and hepatocytes. IGFBP7 expression was also found to inversely correlate with the stages and grade of HCC. Genomic deletion of IGFBP7 was identified in 26% of HCC patients. Forced overexpression of IGFBP7 in AEG-1 overexpressing HCC cells inhibited in vitro growth and induced senescence. When injected into nude mice, in vivo growth was profoundly suppressed, potentially as a result of inhibition of both angiogenesis and IGF1R activation by IGFBP7. Ad.IGFBP7 profoundly inhibited viability and induced apoptosis in multiple human HCC cell lines by inducing Reactive Oxygen Species (ROS) and activating a DNA damage response. N-acetylcysteine could neutralize ROS and rescue the cells from apoptosis. In early phase after Ad.IGFBP7 infection, activation of cell cycle control proteins like Rb, p53, ATM, ATR, CHK1 and CHK2 were identified and G2/M cell cycle arrest was recorded by FACS. Ad.IGFBP7 infection resulted in the activation of p38 MAPK, and a p38 MAPK inhibitor SB 203580 could block the apoptotic process. In orthotopic xenograft models of human HCC in athymic nude mice, intravenous administration of Ad.IGFBP7 profoundly inhibited primary tumor growth and intra-hepatic metastasis. In a nude mouse subcutaneous model, xenografts from human HCC cells were established in both flanks and only left- side tumors received intratumoral injection of Ad.IGFBP7. Ad.IGFBP7 markedly inhibit growth of both left-sided injected tumors and right-sided un- injected tumors by profound suppression of angiogenesis. Conclusion: The present findings provide evidence that IGFBP7 functions as a novel putative tumor suppressor for HCC and establish the corollary that IGFBP7 downregulation can effectively modify AEG-1 function. Targeted overexpression of IGFBP7 may be a potential novel and effective therapy for HCC.

<p>To ensure genomic integrity, dividing cells implement multiple checkpoint pathways during the course of the cell cycle. In response to DNA damage, cells may either halt the progression of the cycle (cell cycle arrest) or undergo apoptosis. This choice depends on the extent of damage and the cell's capacity for DNA repair. Cell cycle arrest induced by double-stranded DNA breaks relies on the activation of the ataxia-telangiectasia (ATM) protein kinase, which phosphorylates cell cycle effectors (e.g., Chk2 and p53) to inhibit cell cycle progression. ATM is an S/T-Q directed kinase that is critical for the cellular response to double-stranded DNA breaks. Following DNA damage, ATM is activated and recruited to sites of DNA damage by the MRN protein complex (Mre11-Rad50-Nbs1 proteins) where ATM phosphorylates multiple substrates to trigger a cell cycle arrest. In cancer cells, this regulation may be faulty and cell division may proceed even in the presence of damaged DNA. We show here that the RSK kinase, often elevated in cancers, can suppress DSB-induced ATM activation in both Xenopus egg extracts and human tumor cell lines. In analyzing each step in ATM activation, we have found that RSK disrupts the binding of the MRN complex to DSB DNA. RSK can directly phosphorylate the Mre11 protein at Ser 676 both in vitro and in intact cells and can thereby inhibit loading of Mre11 onto DSB DNA. Accordingly, mutation of Ser 676 to Ala can reverse inhibition of the DSB response by RSK. Collectively, these data point to Mre11 as an important locus of RSK-mediated checkpoint inhibition acting upstream of ATM activation.</p><p>The phosphorylation of Mre11 on Ser 676 is antagonized by phosphatases. Here, we screened for phosphatases that target this site and identified PP5 as a candidate. This finding is consistent with the fact that PP5 is required for the ATM-mediated DNA damage response, indicating that PP5 may promote DSB-induced, ATM-dependent DNA damage response by targeting Mre11 upstream of ATM.</p> / Dissertation

The p53 tumour suppressor protein lies at the hub of a very complex network of cellular pathways including apoptosis, cell cycle arrest, DNA repair and cellular senescence. However, the mechanism of why and how p53 switches between apoptosis and cell cycle arrest, thereby determining a cell’s fate, remains a mystery to us. To enable us to investigate this ability of p53 to switch between cell cycle arrest and apoptosis, we developed a model which demonstrates similar p53 expression patterns but different functional outcomes. Treating cells with Cisplatin (a common chemotherapeutic drug) and Nutlin-3 (an MDM-2 inhibitor) results in similar high levels of p53 accumulation but different cellular responses. Cisplatin-treated cells undergo apoptosis while Nutlin-treated cells enter cell cycle arrest. Using this model, we explored the localization of p53 and in particular a C-terminal Ser 392 moiety in an attempt to identify how p53 is able to preferentially activate cell cycle arrest or apoptotic pathway.

Objective: To evaluate the anti-tumor and histone deacetylase (HDAC) inhibitory activity of the dietary isothiocyanate, sulforaphane (SFN) in the paediatric cancer,neuroblastoma (NB). Materials and Methods: NB cell line (NUB-7), fibroblasts (FLF; negative control) and MCF-7 (positive control), were treated with SFN for up to 7 days and effects on growth, cytotoxicity, differentiation and tumorigenicity assessed. HDAC inhibition was determined by histone (H3/ H4) acetylation. Results: 10 μM SFN significantly decreased in vitro growth and survival of NUB-7 to 10.22 ± 0.71% (p < 0.001) with no significant effect on FLF. SFN induced G1, G2 and S phase cell cycle arrests and stimulated H3/H4 histone acetylation. SFN markedly decreased NUB-7 clonogenicity and tumorigenicity in vivo. Conclusion: Results suggest that low dose SFN reduces proliferation, survival and tumorigenicity of NB NUB-7. As a dietary factor of negligible intrinsic toxicity SFN is a promising therapeutic agent for the treatment of NB.

Objective: To evaluate the anti-tumor and histone deacetylase (HDAC) inhibitory activity of the dietary isothiocyanate, sulforaphane (SFN) in the paediatric cancer,neuroblastoma (NB). Materials and Methods: NB cell line (NUB-7), fibroblasts (FLF; negative control) and MCF-7 (positive control), were treated with SFN for up to 7 days and effects on growth, cytotoxicity, differentiation and tumorigenicity assessed. HDAC inhibition was determined by histone (H3/ H4) acetylation. Results: 10 μM SFN significantly decreased in vitro growth and survival of NUB-7 to 10.22 ± 0.71% (p < 0.001) with no significant effect on FLF. SFN induced G1, G2 and S phase cell cycle arrests and stimulated H3/H4 histone acetylation. SFN markedly decreased NUB-7 clonogenicity and tumorigenicity in vivo. Conclusion: Results suggest that low dose SFN reduces proliferation, survival and tumorigenicity of NB NUB-7. As a dietary factor of negligible intrinsic toxicity SFN is a promising therapeutic agent for the treatment of NB.

Pomegranate extract (PE) is a standardized whole fruit extract of pomegranate (Punica granatum L.), a fruit with known anti inflammatory and anti cancer properties. In the present study, the effects of PE on two different cell lines of human pancreatic cancer cells, AsPC-1 and PANC-1 were examined. Both cell lines are epithelial cancers but differ morphologically and in their response to treatment with PE. PE inhibited proliferation of both cancer cell lines in a dose dependent and time dependent manner. The reason for this was the potency of PE to cause significant cell cycle arrest in the pancreatic cancer cells, which was noted to be cell specific. AsPC-1 was arrested in G₂ phase while PANC-1 was arrested in G₀/G₁phase of the cell cycle. PE also checked the viability of confluent cancer cells in a concentration dependent manner. In case of AsPC-1 this was due to the action of PE to induce apoptosis, as demonstrated by the Annexin-V assay. In case of PANC-1, a highly invading cancer greatly affected by the presence of surface stem cell markers CD44⁺/CD24⁺, PE was capable of modifying the stem cell markers and induce an increase in the non tumorigenic sub population of cells. A study was also conducted to look at the action of PE in causing inhibition of pancreatic cancer cell proliferation, in comparison to a clinically used chemotherapeutic agent, Paclitaxel. The results indicated that the effect of PE, at clinically feasible doses was superior to that of clinically used doses of Paclitaxel. This data suggests that PE, which is proven to be a safe dietary supplement, looks promising in the prevention and treatment of human pancreatic cancer cells without the side effects of standard drug therapy and could possibly have a role in controlling stem cancer cell population / text