Wild-type viruses with intrinsic oncolytic capacity in human includes DNA viruses like some autonomous parvoviruses and many RNA viruses. Recent advances in molecular biology have allowed the design of several genetically modified viruses, such as adenovirus and herpes simplex virus that specifically replicate in, and kill tumor cells. However, still several hurdles regarding clinical limitations and safety issues should be overcome before this mode of therapy can become of clinical relevance. It includes limited virus spread in tumor masses, stability of virus in the blood, trapping within the liver sinusoids, transendothelial transfer, and/or vector diffusion of viral particles to tumor cells, limited tumor transduction, immune-mediated inactivation or destruction of the virus. For replication-competent vectors without approved antiviral agents, suicide genes might be used as fail-safe mechanism. Cancer stem cells are a minor population of tumor cells that possess the stem cell property of self-renewal. Therefore, viruses that target the defective self-renewal pathways in cancer cells might lead to improved outcomes. In this thesis, data we generated in the field of oncolytic autonomous parvoviruses are presented. We replaced capsid genes by reporter genes and assessed expression in different types of human cancer cells and their normal counterparts, either at the level of whole cell population, (CAT ELISA) or at the single cell level, (FACS analysis of Green Fluorescent Protein). Cat expression was substantial (up to 10000 times background) in all infected tumor cells, despite variations according to the cell types. In contrast, no gene expression was detected in similarly infected normal cells, (with the exception of an expression slightly above background in fibroblasts.). FACS analysis of GFP expression revealed that most tumor cells expressed high level of GFP while no GFP positive normal cells could be detected with the exception of very few (less than 0.1%) human fibroblast cells expressing high level of GFP. We also replace capsid genes by genes coding for the costimulatory molecules B7-1 and B7-2 and show that, upon infection with B7 recombinant virions, only tumor cells display the costimulatory molecules and their immunogenicity was increased without any effect on normal cells. Using a recombinant MVM containig the Herpes Simplex thymidine kinase gene, we could get efficient killing of most tumor cell types in the presence of ganciclovir, whithout affecting normal proliferating cells. We also produced tetracycline inducible packaging cell lines in order to improve recombinant vectors yields. The prospects and limitations of these different strategies will be discussed. An overview is given of the general mechanisms and genetic modifications by which oncolytic viruses achieve tumor cell-specific replication and antitumor efficacy. However, as their therapeutic efficacy in clinical trials is still not optimal, strategies are evaluated that could further enhance the oncolytic potential of conditionally replicating viruses in conjunction with other standard therapies. Another exciting new area of research has been the harnessing of naturally tumor-homing cells as carrier cells to deliver oncolytic viruses to tumors. The trafficking of these tumor-homing cells (stem cells, immune cells and cancer cells), which support proliferation of the viruses, is mediated by specific chemokines and cell adhesion molecules and we are just beginning to understand the roles of these molecules. Finally, we will explore some ways deserving further study in order to be able to utilize various oncolytic viruses for effective cancer treatment.

Gastric cancer is the second leading cause of cancer death worldwide, killing upwards of one million people each year. Apoptosis, a genetically controlled cell death in multicellular eukaryotic organisms, is an important mechanism for embryonic development, immune-system function and maintenance of tissue homeostasis through activation of an intrinsic suicide program to eliminate superfluous, infected, transformed or damaged cells. Single nucleotide polymorphism (SNP) is the most abundant type of genetic variations and is considered to be an important endogenous cause and fundamental factor influencing cancer risk. We conducted a hospital-based case-control study to investigate the association between apoptosis related genes and the risk for gastric cancer. We continuously enrolled 205 patients with pathologically proved gastric cancer and 397 frequency-matched healthy controls at Kaohsiung Veterans General Hospital from 2003 to 2006. Blood derived DNA samples from all participants were genotyped by PCR-RFLP to identify eight SNPs on seven key genes (FAS, FASL, SURVIVIN, XIAP, CASP3, CASP8, CASP9) in apoptotic pathway, but only five SNPs on four genes (FAS, FASL, CASP3, CASP9) were eventually valid for subsequent analyses. Our results showed that significant effects of H. pylori infection, cigarette smoking, alcohol drinking, and consumption of salted food and fermented food on gastric cancer risk while coffee drinking and consumption of vegetables and fruits had significant protective effects against gastric cancer in our study cohort. None of the individual gene polymorphism was associated with the risk of gastric cancer. However, the gene-gene interaction between CASP3 A21926C and CASP9 codon Arg221Gln polymorphisms was significantly associated with gastric cancer risk. In the combined analysis of four apoptosis related genes, our data showed that individuals carrying three or more putative high-risk genotypes were significantly associated with the development of gastric cancer than those who carrying two or less putative high-risk genotypes. Besides, the CASP9 codon Arg221Gln polymorphism was a risk factor for gastric cancer in people equal to or elder than fifty, though not in people younger than fifty. Taken together, our results indicated that SNPs on apoptosis related genes were associated to the development of gastric cancer.

Cellular Inhibitor of Apoptosis 2 (cIAP2) belongs to the IAP family, a group of endogenous proteins that inhibit apoptosis. However, the physiological role of cIAP2 remains poorly defined. Knock-out (KO) and wild type (WT) mice were used to examine the effect of cIAP2 protein on the progression of atherosclerosis in apoE -/- mice. Following the high-fat diet period of 4 and 12 wks, tissues were harvested and analysis focused on the aortic root, the aortic arch, the descending aorta, and the blood. Ex vivo results show a significant decrease in aortic arch lesion area in KO vs. WT in both study groups. Results also show a decrease in aortic root lesion size in KO vs. WT in both study groups. These results support that cIAP2 is an important survival factor for lesion-associated macrophages, since loss of cIAP2 expression in this mouse model reduced atherosclerotic lesion development.

Cellular Inhibitor of Apoptosis 2 (cIAP2) belongs to the IAP family, a group of endogenous proteins that inhibit apoptosis. However, the physiological role of cIAP2 remains poorly defined. Knock-out (KO) and wild type (WT) mice were used to examine the effect of cIAP2 protein on the progression of atherosclerosis in apoE -/- mice. Following the high-fat diet period of 4 and 12 wks, tissues were harvested and analysis focused on the aortic root, the aortic arch, the descending aorta, and the blood. Ex vivo results show a significant decrease in aortic arch lesion area in KO vs. WT in both study groups. Results also show a decrease in aortic root lesion size in KO vs. WT in both study groups. These results support that cIAP2 is an important survival factor for lesion-associated macrophages, since loss of cIAP2 expression in this mouse model reduced atherosclerotic lesion development.

Eukaryotic translation initiation factor 5A (eIF5A) is the only known protein containing the unique amino acid, hypusine. eIF5A is present in all eukaryotic cells and is highly conserved, but its function is not well understood. The present investigation was undertaken to study the regulatory role of eIF5A in the induction of apoptosis in human cancer cell lines. Suppression of eIF5A1 using specific siRNA was shown to have no effect on the growth and proliferation of mammalian cells, although inhibition of the post-translational hypusination of eIF5A1 resulted in G1 phase cell cycle arrest. Treatment of HT-29 human colon adenocarcinoma cells with eIF5A1 siRNA did, however, reduce their sensitivity to pro-apoptotic stimuli including nitric oxide, Actinomycin D, proteaosome inhibition and serum starvation. Furthermore, over-expression of eIF5A1 in HT-29 cells or Hela S3 human cervical carcinoma cells using adenovirus constructs strongly induced apoptosis in a time-dependent manner. The pro-apoptotic effect of eIF5A1 appears to reflect at least in part its ability to activate the mitochondrial pathway of apoptosis in that its up-regulation resulted in dissipation of mitochondrial ∆Ψm, release of cytochrome c, activation of caspase 9 and caspase 3 and translocation of Bax from the cytosol to mitochondria. Similar effects were observed following treatment with eIF5A2, a second isoform of human eIF5A, and in addition eIF5A2 induced up-regulation of cleaved Bcl-2 which is thought to be pro-apoptotic. A mutant of eIF5A1 in which the conserved lysine, lysine50, that is post-translationally modified to hyusine was switched to alanine [eIF5A1(K50A)] also proved capable of inducing apoptosis by activating the mitochondrial pathway. As well, eIF5A1 and eIF5A1(K50A) both induced strong up-regulation of p73, a homolog of the tumor suppressor p53, in Hela S3 cells containing null p53. The finding that up-regulation of eIF5A1 also resulted in activation of caspase 8 indicates that it may be involved in regulation of the death receptor pathway of apoptosis as well. This contention is further supported by confocal microscopy studies indicating that, following its up-regulation, eIF5A1 localizes to the inner surface of the plasma membrane in a time-dependent manner that correlates temporally with the induction of apoptosis. eIF5A1 and its post-translationally modified forms were isolated by 2-dimensional Western blotting and sequenced by mass spectrometry. These analyses indicated that eIF5A1 containing unmodified lysine50 is the form of the protein that accumulates coincident with induction of apoptosis either by up-regulation of eIF5A1 or treatment with NO. These observations, together with the finding that eIF5A1(K50A) is capable of inducing apoptosis, indicate that it is the unhypusinated form of eIF5A1 that is apoptogenic.

Eukaryotic translation initiation factor 5A (eIF5A) is the only known protein containing the unique amino acid, hypusine. eIF5A is present in all eukaryotic cells and is highly conserved, but its function is not well understood. The present investigation was undertaken to study the regulatory role of eIF5A in the induction of apoptosis in human cancer cell lines. Suppression of eIF5A1 using specific siRNA was shown to have no effect on the growth and proliferation of mammalian cells, although inhibition of the post-translational hypusination of eIF5A1 resulted in G1 phase cell cycle arrest. Treatment of HT-29 human colon adenocarcinoma cells with eIF5A1 siRNA did, however, reduce their sensitivity to pro-apoptotic stimuli including nitric oxide, Actinomycin D, proteaosome inhibition and serum starvation. Furthermore, over-expression of eIF5A1 in HT-29 cells or Hela S3 human cervical carcinoma cells using adenovirus constructs strongly induced apoptosis in a time-dependent manner. The pro-apoptotic effect of eIF5A1 appears to reflect at least in part its ability to activate the mitochondrial pathway of apoptosis in that its up-regulation resulted in dissipation of mitochondrial ∆Ψm, release of cytochrome c, activation of caspase 9 and caspase 3 and translocation of Bax from the cytosol to mitochondria. Similar effects were observed following treatment with eIF5A2, a second isoform of human eIF5A, and in addition eIF5A2 induced up-regulation of cleaved Bcl-2 which is thought to be pro-apoptotic. A mutant of eIF5A1 in which the conserved lysine, lysine50, that is post-translationally modified to hyusine was switched to alanine [eIF5A1(K50A)] also proved capable of inducing apoptosis by activating the mitochondrial pathway. As well, eIF5A1 and eIF5A1(K50A) both induced strong up-regulation of p73, a homolog of the tumor suppressor p53, in Hela S3 cells containing null p53. The finding that up-regulation of eIF5A1 also resulted in activation of caspase 8 indicates that it may be involved in regulation of the death receptor pathway of apoptosis as well. This contention is further supported by confocal microscopy studies indicating that, following its up-regulation, eIF5A1 localizes to the inner surface of the plasma membrane in a time-dependent manner that correlates temporally with the induction of apoptosis. eIF5A1 and its post-translationally modified forms were isolated by 2-dimensional Western blotting and sequenced by mass spectrometry. These analyses indicated that eIF5A1 containing unmodified lysine50 is the form of the protein that accumulates coincident with induction of apoptosis either by up-regulation of eIF5A1 or treatment with NO. These observations, together with the finding that eIF5A1(K50A) is capable of inducing apoptosis, indicate that it is the unhypusinated form of eIF5A1 that is apoptogenic.

Altered apoptotic signaling in skeletal muscle has been observed in a number of disease states associated with skeletal muscle atrophy. Therefore, understanding the mechanisms that lead to increased skeletal muscle apoptosis may help to prevent the atrophy associated with various diseases. Apoptosis repressor with caspase recruitment domain (ARC) is a potent anti-apoptotic protein that is able to inhibit apoptosis mediated by both the death-receptor and mitochondrial pathways. In addition, ARC has a unique distribution pattern and is highly expressed in terminally differentiated tissue such as skeletal muscle. To characterize the role of ARC in skeletal muscle morphology and apoptosis, soleus and plantaris muscles of 18 week-old ARC-deficient mice were excised and compared to those of age-matched wild-type littermates. While no differences were seen in muscle weights between genotypes, in the ARC KO animals, the cross-sectional area (CSA) of the soleus was smaller, while the CSA of the plantaris was larger. With respect to fiber type distribution, both muscles demonstrated a shift towards a faster myosin heavy chain expression pattern. For example, soleus muscles of ARC KO animals had significantly less type I fibers and more IIa fibers, while plantaris muscles had significantly less type IIa fibers, and more IIb fibers. In ARC KO animals, type I and IIa fibers were significantly smaller in the soleus, while type IIb fibers were larger in the plantaris. DNA fragmentation (a hallmark of apoptosis) was increased in the soleus, but not plantaris muscles of ARC KO animals. Surprisingly, activity of the proteolytic enzymes caspase-2, -3, -8, and -9, as well as calpains, was not different in either soleus or plantaris muscles. To determine whether a lack of ARC protein affects apoptotic signaling in skeletal muscle, the total expression of pro- and anti-apoptotic proteins were also assessed. In the soleus, no changes were observed in whole tissue AIF, cytochrome c, EndoG, and Smac. In the plantaris, there was no change in total muscle AIF; however, there were trends towards decreased cytochrome c, and increased Smac, as well as a significant decrease in EndoG ARC KO animals. No changes were observed in Bcl-2 and XIAP in the soleus; however, there were significant reductions in FLIP(s) and HSP70 content. In the plantaris, no changes were observed in anti-apoptotic protein content. Subcellular fractionation of red quadriceps for ARC KO mice revealed an increased Bax:Bcl-2 ratio in the isolated mitochondrial fractions. Furthermore, in cytosolic fractions of red quadriceps, AIF protein content was significantly increased in ARC KO animals. Conversely, no changes in apoptotic-related protein content were observed in any fractions from white quadriceps between groups. In agreement with these findings, isolated mitochondria from ARC-deficient animals were more susceptible to calcium induced swelling, as well as membrane potential loss compared to controls. Taken together, these results suggest that in slow-oxidative skeletal muscle of ARC-deficient mice there is increased apoptosis due to caspase-independent, mitochondrial-mediated apoptotic signaling. Furthermore, this study is the first to show ARC plays an important role in skeletal muscle morphology, as ARC KO mice have an altered skeletal muscle phenotype and morphology.

ROGDI was a novel gene with unknown function, located on human chromosome 16p13.3. The coding region of the gene is 864 bp that encodes 287 amino acids. According to GenBank database, ROGDI contains leucine zipper domain. Previous studies in our laboratory showed that ROGDI increases cell proliferation in cell lines. In addition, overexpression of ROGDI induces p53 and p27 mRNA levels in human glioma cell line T98G and U251. In this study, we use two hepatocellular carcinoma cell lines, Hep G2 and Hep 3B, which contains wild-type and deleted tumor suppressor protein p53 respectively to investigate the expression of p53 and the response of anticancer drugs treatment in ROGDI overexpression cells. In addition, we compare the relation between the cell apoptosis the expression of p53 and ROGDI. Hence, we found that expression of p53 and ROGDI influences the cell response to anticancer drugs and induces apoptosis.

Four small ubiquitin-like modifier (SUMO) isoforms termed SUMO-1, -2, -3 and -4 have been identified in human. Most SUMO-1/2 proteins are localized in nucleus, whereas SUMO-1 protein exhibits 44% homolog with SUMO-2 protein. Over 50 proteins have been identified as the target proteins for SUMO-1 modification and these include transcription factors, their cofactors, regulators, nuclear body proteins, nuclear pore complex proteins, DNA repair proteins, and viral proteins. However, only a handful of SUMO-2 targets are known and SUMO-2 modification may response to environmental stress. SUMO-1 may interact with Fas/APO-1 and TNF receptor 1 on yeast two hybrid interactions; however, it is not clear whether SUMO would enhance apoptosis or response to biological stress. Helicobacter pylori (H. pylori) defined as a gastric carcinogen is definite a biological stress to the cells. It causes gastric epithelial cell damage by apoptosis. In this study whether the SUMO-1/2 pathway constitutes an element of the cellular response to the H. pylori infection was examined. Overexpression of SUMO-1/2 for 12 hours had no effects on the apoptotic activities of cells; however it enhanced apoptosis during H. pylori infection. Overexpression of SUMO-1/2 for 48 hours increased the apoptosis of cells; however only SUMO-2 enhanced apoptosis significantly during H. pylori infection. The enhancements are more powerful for SUMO-2 than that of SUMO-1. Inactive SUMO, a cytoplasm dispersed sumoylation-incompetent mutant, eliminates such activities, suggesting that sumoylation or SUMO interactions are involved in the apoptotic enhancement. The percentages of cells with cytoplasmic SUMO-2 were increased 22% by H. pylori infection for 2 hours and SUMO-1 were increased 11%. The translocalization of SUMO-1 was blocked by leptomycin B; however, it did not work on SUMO-2. Leptomycin B could also inhibit SUMO-1 enhanced apoptosis during H. pylori infection, whereas it had no effects on SUMO-2. It is concluded that SUMO-1/2 pathway constitutes an element of the cellular response to H. pylori infection by enhancing apoptosis through shuttling from nucleus to cytoplasm. SUMO-1 is via a CRM1-dependent pathway while SUMO-2 is via a CRM1-independent pathway.

The pathological hallmarks of Alzheimer¡¦s disease (AD) include neurofibrillary tangle, amyloid plaques and neuronal cell death. Neurofibrillary tangle is composed of hyperphosphorylated tau whereas APP (