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This study investigated the spatial distributions of mortality rates of six cancers: mesothelioma, lung cancer, intestinal cancer, nasopharyngeal and laryngeal cancer, liver cancer, and stomach cancer in Dayao using Geographic Information Systems. Relationships between the mortality rates of the six cancers and land use patterns were investigated by Pearson Correlation Coefficients. The results indicated that the mortality rates of nasopharyngeal and laryngeal cancer, lung cancer, intestinal cancer, and mesothelioma were significantly associated with outcropped asbestos. Both the proportions of farmland and urban area were positively related to the mortality rates of nasopharyngeal and laryngeal cancer, lung cancer, intestinal cancer, and mesothelioma, and significant negative correlations were found between the proportion of forestland and nasopharyngeal and laryngeal cancer and intestinal cancer. It can be concluded that naturally occurring asbestos may significantly elevate the mortality rates of nasopharyngeal and laryngeal cancer, intestinal cancer, lung cancer, and mesothelioma. Moreover, higher proportions of farmland, urban area, and lower proportions of forested land may elevate the mortality rate of the four cancers.

Cadmium (Cd)-contaminated rice is one of the most important sources of cadmium exposure in the general population from some Asian countries. This study was conducted to assess cadmium exposure from uncooked rice in rural mining areas based on the bioaccessible fraction of cadmium using an in vitro digestion model. The biotoxic effects of cadmium in uncooked rice from mining areas were much higher than those in the control area, based not only on their higher total concentration (52.49 vs. 7.93 μg kg(-1)), but also on their higher bioaccessibility (16.94% vs. 2.38%). In the mining areas, the bioaccessible fraction of cadmium in uncooked rice has a significant positive correlation with the total concentration of cadmium in rice and there was quarterly unsafe rice to the public in the mining areas. The results indicated that the in vitro digestion model could be a useful and economical tool for providing the solubilization or bioaccessibility of uncooked rice in the mining area. The results could be helpful in conducting future experiments of cooked rice in the vitro model.

Phosphorylation at glutamate receptor subunit 1(GluR1) Ser845 residue has been widely accepted to involve in GluR1-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking, but the in vivo evidence has not yet been established. One of the main obstacles is the lack of effective methodologies to selectively target phosphorylation at single amino acid residue. In this study, the Escherichia coli-expressed glutathione-S-transferase-tagged intracellular carboxyl-terminal domain of GluR1 (cGluR1) was phosphorylated by protein kinase A for in vitro selection. We have successfully selected aptamers which effectively bind to phospho-Ser845 cGluR1 protein, but without binding to phospho-Ser831 cGluR1 protein. Moreover, pre-binding of the unphospho-cGluR1 protein with these aptamers inhibits protein kinase A-mediated phosphorylation at Ser845 residue. In contrast, the pre-binding of aptamer A2 has no effect on protein kinase C-mediated phosphorylation at Ser831 residue. Importantly, the representative aptamer A2 can effectively bind the mammalian GluR1 that inhibited GluR1/GluR1-containing AMPA receptor trafficking to the cell surface and abrogated forskolin-stimulated phosphorylation at GluR1 Ser845 in both green fluorescent protein-GluR1-transfected human embryonic kidney cells and cultured rat cortical neurons. The strategy to use aptamer to modify single-residue phosphorylation is expected to facilitate evaluation of the potential role of AMPA receptors in various forms of synaptic plasticity including that underlying psychostimulant abuse.

Purpose: This study aimed to develop an efficient and safe strategy to introduce suicide genes into colon cancer cells. Experimental Design: In this study, we fused an enhanced carcinoembryonic antigen promoter (CEA) to a suicide gene, cytosine deaminase (CD). This construct was delivered into colon cancer cells using calcium phosphate nanoparticles (CPNP). The cells were then treated with the prodrug 5-FC. The therapeutic effect was evaluated in vitro and in vivo. Results: Our study showed that the CEA promoter–driven, CPNP-delivered suicide gene was only expressed in CEA-positive colon cancer cells, and resulted in significant cytotoxicity after administration of the prodrug 5-FC in vitro. Moreover, our in vivo study showed that CPNP-mediated CEA-CD delivery, together with 5-FC treatment, resulted in significant tumor growth delay in xenograft human colon carcinoma. Conclusions: Our study indicates that the combination of CPNP and CEA-CD gene expression represents a novel approach for CEA-positive tumor gene therapy.

We designed and tested, in vitro, an adenoviral construct containing the feline interleukin-12 (IL-12) gene under control of the heat-inducible promoter HSP70B. This construct, AdhspfIL12, was used in a phase I trial in feline soft tissue sarcomas. During the course of our experiments, we noted that IL-12 was being produced in the transfected Crandell Feline Kidney (CrFK) cells under certain conditions even in the absence of hyperthermia. This observation was further explored to identify the cause of this unintended HSP70B induction.We used real-time PCR as a sensitive method to quantitatively detect the presence of even small amounts of IL-12 mRNA. This served as a surrogate indicator of HSP70B induction. Various conditions were tested to induce the heat shock promoter, including nutritional deprivation, radiation and changes in pH.Nutritional stresses, specifically the absence of glucose and glutamine, could induce the heat shock promoter, thus, resulting in production of the downstream gene product. Other factors known to trigger the heat shock response, pH change, and reactive oxygen species production were also studied but were not found to contribute to heat shock promoter induction in our setting.The human heat shock promoter (HSP70B) is reported to be an efficient and tightly regulated promoter. We discovered, using sensitive real-time PCR techniques, that it can also be induced in response to cellular nutrient stresses. The pros and cons of this phenomenon and its implications for cancer gene therapy are discussed.

Aptamers offer advantages over other oligonucleotide-based approaches that artificially interfere with target gene function due to their ability to bind protein products of these genes with high affinity and specificity. However, RNA aptamers are limited in their ability to target intracellular proteins since even nuclease-resistant aptamers do not efficiently enter the intracellular compartments. Moreover, attempts at expressing RNA aptamers within mammalian cells through vector-based approaches have been hampered by the presence of additional flanking sequences in expressed RNA aptamers, which may alter their functional conformation. In this report, we successfully expressed a 'pure' RNA aptamer specific for NF-kappaB p50 protein (A-p50) utilizing an adenoviral vector employing the H1 RNA polymerase III promoter. Binding of the expressed aptamer to its target and subsequent inhibition of NF-kappaB mediated intracellular events were demonstrated in human lung adenocarcinoma cells (A549), murine mammary carcinoma cells (4T1) as well as a human tumor xenograft model. This success highlights the promise of RNA aptamers to effectively target intracellular proteins for in vitro discovery and in vivo applications.

DNA fragmentation is a hallmark of apoptosis (programmed cell death). However, the biological function of apoptotic DNA fragmentation remains unclear. Here, we show that DNA fragmentation factor plays an important role for maintaining genomic stability. Inhibition or loss of the DNA fragmentation factor (DFF)/caspase-activated DNase (CAD), whose nuclease activity is responsible for digesting genomic DNA during apoptosis, led to significant increases in spontaneous or induced gene mutations, gene amplifications, and chromosomal instability in primary mouse cells and transformed human cell lines. The mechanism underlying genetic instability in DFF/CAD-deficient cells, at least in part, involves a small but significant elevation in the survival of cells exposed to ionizing radiation, suggesting that apoptotic DNA fragmentation factor contributes to genomic stability by ensuring the removal of cells that have suffered DNA damage. In support of this hypothesis are the observations of increased cellular transformation of mouse embryonic cells from the DFF/CAD-null mice and significantly enhanced susceptibility to radiation-induced carcinogenesis in these mice. These data, in combination with published reports on the existence of tumor-specific gene mutations/deletions in the DFF/CAD genes in human cancer samples, suggest that apoptotic DNA fragmentation factor is required for the maintenance of genetic stability and may play a role in tumor suppression.

GW112 is a novel gene that has little homology to other known genes. It is overexpressed in a number of human tumor types, especially in those of the digestive system. We show here that GW112 is associated with GRIM-19, a protein known to be involved in regulating cellular apoptosis. Functionally, GW112 could significantly attenuate the ability of GRIM19 to mediate retinoic acid-IFN-β-mediated cellular apoptosis and apoptosis-related gene expression. In addition, GW112 demonstrated strong antiapoptotic effects in tumor cells treated with other stress exposures such as hydrogen peroxide. Finally, forced overexpression of GW112 in murine prostate tumor cells led to more rapid tumor formation in a syngeneic host. Taken together, our data suggest that GW112 is an important regulator of cell death that plays important roles in tumor cell survival and tumor growth.

Purpose: To develop a novel conditionally replicative adenovirus vector that targets telomerase-positive cancer cells. Experimental Design: A telomerase gene-derived promoter was used to control the expression of the E1a gene so that the E1a gene is only expressed in telomerase-positive tumor cells. In addition, a reporter gene was also engineered into the vector so that its infection and replication can be monitored easily. Results: A novel recombinant adenovirus vector that could selectively replicate in telomerase-positive cancer cells was made successfully. This vector showed active replication in a panel of cancer cells and minimal replication in normal human fibroblast or epithelial cells. The recombinant vector could effectively lyse various cultured tumor cells even at very low multiplicity of infection. The replication efficiency in tumor cells is over 103-fold more than normal fibroblast and epithelial cells. In s.c. tumor models, the newly developed telomerase-selective adenovirus vectors exhibited significantly more virus replication and reporter gene expression. Conclusions: The telomerase-targeted adenovirus vector has significant potential as an oncolytic virus as well as a tumor-specific therapeutic gene delivery vehicle.

An efficient approach to the large-scale production of rAAV will significantly facilitate the application of this promising gene delivery vector in human gene therapy applications. In this study, we report a novel approach to rAAV production that is based exclusively on the adenovirus vector, without the need for plasmid transfections and special packaging cell lines. All components required for rAAV production, including the rep and cap genes, and the therapeutic gene(s) are delivered to the widely used 293 packaging cell line by adenovirus vectors. High-titer rAAV vectors (200-600 infectious units/producer cell) were obtained by use of this approach. As adenovirus vectors can be produced to high titers and they can infect cells in suspension efficiently, this approach may be amenable to scaled-up production of rAAV vectors.

Background and purpose Patients with life-threatening illnesses, such as cancer, experience emotional distress. This study was to investigate the molecular and cellular mechanisms of relevant psychological stressor on tumor growth and therapeutic resistance. Materials and Methods Stress was induced in C57BL/6J mice bearing LLC lung tumors by exposure to a conspecific mice receiving inescapable foot shocks. Mice were irradiated at 7 Gy for 3 consecutive days. Behaviors were monitored by open field test (OFT), elevated plus maze (EPM), sucrose preference test (SPT), and learned helplessness (LH) test. Protein expression in tissues and cultured cells were measured by Western blot. Results This study in animals showed that observing a conspecific mouse receiving foot shocks induced depression like behaviors with increased plasma corticosterone and adrenaline levels which increased tumor growth and radioresistance. Stress increased Wnt1, Drosha, and vimentin expression and decreased E-cadherin expression in tumor tissues. The combination of stress and irradiation enhanced radioresistance along with the increase in vimentin expression. The in vitro study showed that a β2-adrenergic receptor (β2-AR) agonist blocked irradiation-induced cell apoptosis and decreased cell viability, while silencing β2-AR expression reduced the protective effects of β2-AR agonist. β2-AR agonist obviously increased Wnt1 and Drosha expression in LLC-1 cells. Conclusion Psychological stress increased tumor growth and enhanced radioresistance associated with the activation of epithelial-mesenchymal transition by stress hormone-stimulated adrenergic receptors.

Our recent studies have demonstrated that integrin-linked kinase (ILK) is involved in the induction and maintenance of cocaine behavioral sensitization and chronic cocaine-induced neural plasticity in the nucleus accumbens (NAc) core. In the present study, we used ILK silencing to investigate how ILK may influence cocaine-induced neural plasticity. Adeno-associated virus carrying a small interfering RNA-ILK cassette under the control of an inducible Tet-On system was injected into the NAc core of Sprague-Dawley rats. Induced silencing was established during repeated cocaine injections (sensitization induction period) or between withdrawal days 9 and 22 (sensitization maintenance period). Under both paradigms, established cocaine sensitization under non-silenced conditions was associated with enhanced PSD-95 and synapsin I protein expression as well as enhanced Ser(845) phosphorylation of the GluR1 subunit on withdrawal day. Silencing ILK expression under both paradigms prevented or reversed these changes. Importantly, ILK appears to form a complex with PSD-95 and synapsin I because it co-immunoprecipitated with each of these proteins. Together, these data suggest that ILK exerts pleiotropic actions by regulating pre- and postsynaptic neural plasticities within the NAc core in response to repeated cocaine exposure.

Due to the prevalence of tumor chemoresistance, the clinical response of advanced non-small cell lung cancer (NSCLC) to chemotherapy is poor. We suppressed tumor resistance to doxorubicin (Dox) in A549 cells, a human NSCLC cell line, both in vitro and in vivo in a lung tumor xenograft model, using a novel adenoviral expression system to deliver an RNA aptamer (A-p50) that specifically inhibits nuclear factor-kappaB (NF-kappaB) activation. By achieving selective, targeted, and early inhibition of NF-kappaB activity, we demonstrate that NF-kappaB plays a critical role in Dox-induced chemoresistance by regulating genes involved in proliferation (Ki-67), response to DNA damage (GADD153), antiapoptosis (Bcl-XL), and pH regulation (CA9). This Dox-induced NF-kappaB activation and subsequent chemoresistance is dependent on expression of p53. We also demonstrate that NF-kappaB promotes angiogenesis in the presence of Dox via the hypoxia-inducible factor-1alpha/vascular endothelial growth factor (HIF-1alpha/VEGF) pathway, revealing a previously unknown mechanism of NSCLC resistance to Dox. These studies provide important insights into the mechanisms of Dox-induced chemoresistance, and they demonstrate a novel, effective, and clinically practical strategy for interfering with these processes.

Prolonged activation of NF-kappaB is involved in the pathogenesis of chronic inflammatory diseases and associated cancers. NF-kappaB activation is considered to be a main mechanism opposing TNFalpha-induced apoptosis. We investigated whether inhibition of NF-kappaB could sensitize tumor and endothelial cells to TNFalpha-induced apoptosis. As such, we developed a novel H1 RNA polymerase III promoter driven adenoviral vector to express an RNA aptamer, Ad-A-p50, which selectively inhibits NF-kappaB activation in the nucleus. This event sensitizes human lung adenocarcinoma cells (A549) and human endothelial cells (HUVEC) to TNFalpha-induced apoptosis through the multiple pathways regulated by NF-kappaB, including Bcl-XL, HIF-1alpha, and VEGF. Our findings also suggest a new mechanism of HIF-1alpha regulation by NF-kappaB in the normoxic environment. RNA aptamer inhibition of NF-kappaB offers exciting opportunities for sensitizing inflammatory and tumor cells to TNFalpha-induced apoptosis.

Interleukin-12 (IL-12) is a pro-inflammatory cytokine that has shown great promise as a therapeutic agent in experimental models of infectious disease and cancer. However, it is also a highly toxic molecule and for that reason has not been accepted readily into the clinic. A replication-deficient adenoviral vector was designed to deliver the feline interleukin-12 gene into tumour cells. The interleukin-12 gene has been placed under control of a heat inducible promoter, human heat shock promoter 70b, with the intent of spatially and temporally controlling the expression of IL-12, thus limiting its toxicity. In vitro, the transfection efficiency of the adenoviral vector, the effect of multiplicity of infection and the production of biologically active feline IL-12 were studied in the infected cells in response to a range of temperatures. This adenoviral vector will be a useful tool to examine the effects of intra-tumoural IL-12 delivery in a spontaneously occurring feline soft tissue sarcoma model.

Alphavbeta3 integrin is a crucial factor involved in a variety of physiological processes, such as cell growth and migration, tumor invasion and metastasis, angiogenesis, and wound healing. Alphavbeta3 integrin exerts its effect by regulating endothelial cell (EC) migration, proliferation, and survival. Inhibiting the function of alphavbeta3 integrin, therefore, represents a potential anti-cancer, anti-thrombotic, and anti-inflammatory strategy. In this study, we tested an RNA aptamer, Apt-alphavbeta3 that binds recombinant alphavbeta3 integrin, for its ability to bind endogenous alphavbeta3 integrin on the surface of cells in culture and to subsequently affect cellular response. Our data illustrate that Apt-alphavbeta3 binds alphavbeta3 integrin expressed on the surface of live HUVECs. This interaction significantly decreases both basal and PDGF-induced cell proliferation as well as inhibition of cell adhesion. Apt-alphavbeta3 can also reduce PDGF-stimulated tube formation and increase HUVEC apoptosis through inhibition of FAK phosphorylation pathway. Our results demonstrate that by binding to its target, Apt-alphavbeta3 can efficiently inhibit human EC proliferation and survival, resulting in reduced angiogenesis. It predicts that Apt-alphavbeta3 could become useful in both tumor imaging and the treatment of tumor growth, atherosclerosis, thrombosis, and inflammation.

Hypoxia-inducible factor-1alpha (HIF-1alpha) is an important transcriptional factor that is activated when mammalian cells experience hypoxia, a tumor microenvironmental condition that plays pivotal roles in tumor progression and treatment. In this study, we examined the idea of down-regulating HIF-1alpha in tumor cells for therapeutic gain. We show that the expression levels of HIF-1alpha can be significantly attenuated by use of the recently established small interfering RNA technology in combination with adenovirus-mediated gene transfer. Down-regulation of the HIF-1alpha protein enhanced hypoxia-mediated tumor cell apoptosis in vitro. Subcutaneous tumor growth was also prevented from cells with attenuated HIF-1alpha expression. In addition, intratumoral injection of adenovirus encoding the HIF-1alpha-targeted small interfering RNA had a small but significant effect on tumor growth when combined with ionizing radiation. Therefore, our results provide proof of HIF-1alpha as an effective target for anticancer therapy. They also suggest that an adenovirus-based small interfering RNA gene transfer approach may be a potentially effective adjuvant strategy for cancer treatment.

Integrin-linked kinase (ILK) is a serine-threonine kinase associated with anchorage-independent growth and tumorigenic transformation. Previous studies indicate that overexpression of ILK is common among several types of tumors, and it is involved in the regulation of tumor cell survival under stress. In this study, we examined the effects of ILK expression on tumor cellular response to hyperthermia.We used an adenovirus-mediated approach to overexpress the ILK gene in a prostate cancer cell line and examine its effects on heat stress-induced cell death. Clonogenic survival, as well as apoptosis, was evaluated in cells that overexpress ILK. In addition, the ability to form tumors in vivo was examined in syngeneic hosts. Finally, potential molecular mechanisms of ILK-mediated resistance to heat were examined by determining the status of a variety of signal transduction pathways.ILK overexpression made tumor cells significantly more resistant to the cell-killing effects of hyperthermia. This was correlated at the molecular level with the down-regulation of hyperthermia-induced activation of stress-activated protein kinase/c-Jun-NH(2)-terminal kinase, p38 mitogen-activated protein kinase activities, and caspase 9. The overexpression of ILK was also shown to induce a more rapid tumor growth in a murine prostate cancer cell lineILK plays an important role in tumor growth and tumor response to hyperthermia treatment.

Background Integrin-linked kinase (ILK) is a protein that plays important roles in extracellular matrix-mediated signaling. It has been shown that ILK is expressed preferentially in cardiac and skeletal muscles. Evidence points to its role as an upstream regulator of protein kinase B, a critical player in apoptosis. Because oxidized LDL (oxLDL) is thought to promote atherogenesis by causing the apoptosis of endothelial cells, we investigated the potential roles that ILK may play in oxLDL-induced apoptosis in vascular endothelial cells. Methods and Results Transcriptional and translational levels of ILK were investigated with reverse-transcriptase polymerase chain reaction and Western analysis. oxLDL treatment induced both the transcription and the translation of the ILK gene in endothelial cells. A recombinant adenovirus vector encoding the ILK gene was constructed to investigate its potential role in oxLDL-induced apoptosis in human umbilical vein endothelial cells and mouse lymphoid vein endothelial cells transformed by simian virus 40. In both types of cells, overexpression of the ILK gene significantly prevented oxLDL-induced apoptosis or cell death, as evaluated by 2 independent assay methods. Furthermore, we showed that ILK could inhibit oxLDL-induced upregulation of the kinase activity of p38 mitogen-activated protein kinase, which is often associated with stress-induced pro-apoptotic signal transduction. Finally, examination of other factors, such as bcl-2, bcl-xl, caspase 3, and caspase 9, demonstrated significant changes that were correlated with oxLDL treatment and ILK overexpression. Conclusion ILK may be an important factor involved in the regulation of oxLDL-induced apoptosis in vascular endothelial cells. Modifying its activity may be a useful approach for prevention of endothelial cell injury in oxLDL-induced atherosclerosis.