Adenovirus-mediated Transfer Research Articles

Short-Term Overexpression of a Constitutively Active Form of AMP-Activated Protein Kinase in the Liver Leads to Mild Hypoglycemia and Fatty Liver

AMP-activated protein kinase (AMPK) is a major therapeutic target for the treatment of diabetes. We investigated the effect of a short-term overexpression of AMPK specifically in the liver by adenovirus-mediated transfer of a gene encoding a constitutively active form of AMPKalpha2 (AMPKalpha2-CA). Hepatic AMPKalpha2-CA expression significantly decreased blood glucose levels and gluconeogenic gene expression. Hepatic expression of AMPKalpha2-CA in streptozotocin-induced and ob/ob diabetic mice abolished hyperglycemia and decreased gluconeogenic gene expression. In normal mouse liver, AMPKalpha2-CA considerably decreased the refeeding-induced transcriptional activation of genes encoding proteins involved in glycolysis and lipogenesis and their upstream regulators, SREBP-1 (sterol regulatory element-binding protein-1) and ChREBP (carbohydrate response element-binding protein). This resulted in decreases in hepatic glycogen synthesis and circulating lipid levels. Surprisingly, despite the inhibition of hepatic lipogenesis, expression of AMPKalpha2-CA led to fatty liver due to the accumulation of lipids released from adipose tissue. The relative scarcity of glucose due to AMPKalpha2-CA expression led to an increase in hepatic fatty acid oxidation and ketone bodies production as an alternative source of energy for peripheral tissues. Thus, short-term AMPK activation in the liver reduces blood glucose levels and results in a switch from glucose to fatty acid utilization to supply energy needs.

The effects of the overexpression of recombinant uncoupling protein 2 on proliferation, migration and plasminogen activator inhibitor 1 expression in human vascular smooth muscle cells

Increased oxidative stress in vascular smooth muscle cells (VSMCs) has been implicated in the pathogenesis of accelerated atherosclerosis in patients with diabetes mellitus. Uncoupling protein 2 (UCP-2) is an important regulator of intracellular reactive oxygen species (ROS) production. We hypothesised that UCP-2 functions as an inhibitor of the atherosclerotic process in VSMCs. Overexpression of human UCP-2 was performed in primary cultured human VSMCs (HVSMCs) via adenovirus-mediated gene transfer. Its effects on ROS production, AP-1 activity, plasminogen activator inhibitor 1 (PAI-1) gene expression, and cellular proliferation and migration were measured in response to high glucose and angiotensin II (Ang II) concentrations, two major factors in the pathogenesis of atherosclerosis in patients with diabetes and hypertension. Mitochondrial membrane potential and NAD(P)H oxidase activity were also measured. High glucose and Ang II caused transient mitochondrial membrane hyperpolarisation. They also significantly stimulated ROS production, NAD(P)H oxidase activity, mitochondrial membrane potential, AP-1 activity, PAI-1 mRNA expression, and proliferation and migration of HVSMCs. Adenovirus-mediated transfer of the UCP-2 gene reversed all of these effects. The present study demonstrates that UCP-2 can modify atherosclerotic processes in HVSMCs in response to high glucose and Ang II. Our data suggest that agents increasing UCP-2 expression in vascular cells may help prevent the development and progression of atherosclerosis in patients with diabetes and hypertension.

Telomerase Mediates Vascular Endothelial Growth Factor-dependent Responsiveness in a Rat Model of Hind Limb Ischemia

Telomere dysfunction contributes to reduced cell viability, altered differentiation, and impaired regenerative/proliferative responses. Recent advances indicate that telomerase activity confers a pro-angiogenic phenotype to endothelial cells and their precursors. We have investigated whether telomerase contributes to tissue regeneration following hind limb ischemia and vascular endothelial growth factor 165 (VEGF(165)) treatment. VEGF delivery induced angiogenesis and increased expression of the telomerase reverse transcriptase (TERT) and telomerase activity in skeletal muscles and satellite and endothelial cells. Adenovirus-mediated transfer of wild type TERT but not of a dominant negative mutant, TERTdn, significantly induced capillary but not arteriole formation. However, when co-delivered with VEGF, TERTdn abrogated VEGF-dependent angiogenesis, arteriogenesis, and blood flow increase. This effect was paralleled by in vitro evidence that telomerase inhibition by 3'-azido-3'-deoxythymidine in VEGF-treated endothelial cells strongly reduced capillary density and promoted apoptosis in the absence of serum. Similar results were obtained with adenovirus-mediated expression of TERTdn and AKTdn, both reducing endogenous TERT activity and angiogenesis on Matrigel. Mechanistically, neo-angiogenesis in our system involved: (i) VEGF-dependent activation of telomerase through the nitric oxide pathway and (ii) telomerase-dependent activation of endothelial cell differentiation and protection from apoptosis. Furthermore, detection of TERT in activated satellite cells identified them as VEGF targets during muscle regeneration. Because TERT behaves as an angiogenic factor and a downstream effector of VEGF signaling, telomerase activity appears required for VEGF-dependent remodeling of ischemic tissue at the capillaries and arterioles level.

Adenovirus-Mediated Transfer and Overexpression of Heme Oxygenase 1 cDNA in Lungs Attenuates Elastase-Induced Pulmonary Emphysema in Mice

Heme oxygenase 1 (HO-1) is an inducible enzyme that catalyzes heme to generate bilirubin, ferritin, and carbon monoxide. Because enhanced expression of HO-1 provides an anti-inflammatory effect and confers cytoprotection, we examined whether HO-1 overexpression induced by inoculation of mice with an adenovirus encoding HO-1 (Ad.HO-1) in the lung would prevent pulmonary emphysema induced by porcine pancreatic elastase (PPE). Pretreatment with Ad.HO-1, which upregulated production of HO-1 in the lung, attenuated the PPE-induced increase of neutrophils in bronchoalveolar lavage fluid (BALF) and enlargement of alveoli. It also reduced PPE-induced elevated levels of tumor necrosis factor alpha, interleukin (IL)-6, and keratinocyte-derived chemokine, and increased the level of anti-inflammatory cytokine IL-10 in BALF. These results suggest that Ad.HO-1-induced HO-1 overexpression suppressed PPE-induced emphysema by attenuating neutrophilic inflammation via modulating cytokine and chemokine profiles in mouse lungs.

Increased fluid secretion after adenoviral-mediated transfer of the human aquaporin-1 cDNA to irradiated miniature pig parotid glands

The treatment of most head and neck cancer patients includes ionizing radiation (IR). Salivary glands in the IR field suffer irreversible damage. Previously, we reported that adenoviral (Ad)-mediated transfer of the human aquaporin-1 (hAQP1) cDNA to rat submandibular glands following IR restored salivary flow to near normal levels. It is unclear if this strategy is useful in larger animals. Herein, we evaluated AdhAQP1-mediated gene transfer after parotid gland IR (20 Gy) in the miniature pig. Sixteen weeks following IR, salivation from the targeted gland was decreased by >80%. AdhAQP1 administration resulted in a dose-dependent increase in parotid salivary flow to approximately 80% of pre-IR levels on day 3. A control Ad vector was without significant effect. The effective AdhAQP1 dose was 2.5 x 10(5) pfu/microl infusate, a dose that leads to comparable transgene expression in murine and minipig salivary glands. Three days after Ad vector administration little change was observed in clinical chemistry and hematology values. These findings demonstrate that localized delivery of AdhAQP1 to IR-damaged salivary glands increases salivary secretion, without significant general adverse events, in a large animal model.

Adenovirus-Mediated Transfer of Human Placental Ectonucleoside Triphosphate Diphosphohydrolase to Vascular Smooth Muscle Cells Suppresses Platelet Aggregation In Vitro and Arterial Thrombus Formation In Vivo

Platelet-rich thrombus formation is a critical event in the onset of cardiovascular disease. Because ADP plays a significant role in platelet aggregation, its metabolism is important in the regulation of platelet activation and recruitment. Ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) is a key enzyme involved in vascular ADP metabolism. We recently isolated 2 isoforms of E-NTPDase from the human placenta. The present study examined whether these isoforms suppress platelet aggregation and thrombus formation after adenovirus-mediated gene transfer to vascular smooth muscle cells (SMCs). We constructed adenovirus vectors expressing human placental E-NTPDase isoforms I (AdPlac I) and II (AdPlac II) or bacterial beta-galactosidase (AdLacZ). Vascular SMCs infected with AdPlac I expressed significant NTPDase activity and inhibited the platelet aggregation induced by ADP and collagen in vitro. In contrast, SMCs infected with AdPlac II and AdLacZ did not exert antiplatelet effects. To investigate the antithrombotic and antiproliferative effects of placental E-NTPDase isoform I in vivo, we generated thrombosis in rat carotid arteries by systemically administered rose Bengal and transluminal green light 5 days after gene transfer and examined neointimal growth 3 weeks after thrombus formation. Blood flow in AdLacZ-infected arteries rapidly deteriorated and vanished within 96+/-18 seconds of occlusive thrombus formation. In contrast, blood flow in AdPlac I-infected arteries was preserved for at least 10 minutes during irradiation. In addition, thrombus formation and subsequent neointimal growth were obviously suppressed. The local expression of placental E-NTPDase in injured arteries might prevent arterial thrombosis and subsequent neointimal growth.

Inhibition of proliferation of human lens epithelial cell line HLE-B3 by adenovirus-mediated transfer of antisense c-myc gene

To investigate the effects of adenovirus-mediated transfer of antisense c-myc gene on proliferation and survival of human lens epithelial cell. Immortalized human lens epithelial cell line (HLE-B3) was infected by replication-deficient adenovirus of antisense c-myc gene (Ad-AS-myc). The expression of c-myc mRNA and protein was detected by RT-PCR and Western blotting respectively. Cell proliferation was analyzed by cell growth curve and MTT colorimetric assay, cell death and cell cycle of HLE-B3 were examined by flow cytometry. More than 80% of HLE-B3 cells were infected successfully after 48 hours incubation with Ad-AS-myc adenovirus. The expression of c-myc mRNA and protein was decreased. Ad-AS-myc suppressed the growth rate, enhanced the cell death (18.33% after 48 hours and 26.93% after 96 hours transduction). The proportions of G1 phase cells were increased to (60.50 +/- 7.59)% after 48 hours and (81.90 +/- 8.60)% after 96 hours transduction, while the proportions of S phase cells were decreased to (28.40 +/- 3.38)% after 48 hours and (16.75 +/- 8.97)% after 96 hours infection. Adenovirus-mediated antisense c-myc may inhibit proliferation and induce apoptosis of HLE-B3.

Inhibition of human lens epithelial B-3 cell proliferation by adenovirus-mediated transfer of antisense c-myc construct

To investigate the effects of adenovirus-mediated transfer of antisense c-myc construct on human lens epithelial B-3 (HLE B-3) cell proliferation, apoptosis and cell cycle. HLE B-3 cell cultures were transduced with replication-defective adenovirus bearing either a nuclear-targeted beta-galactosidase (Ad-lacZ) or an antisense c-myc construct (Ad-AS-myc). The presence of beta-galactosidase activity in the transduced cultures was detected by immunohistochemical X-Gal staining, while c-myc mRNA and protein expression levels were evaluated by RT-PCR and Western blot analysis. HLE B-3 cell proliferation within 96 h after the transduction was analyzed by cell counting and MTT colorimetric assay. Apoptosis and cell cycle of the HLE-B3 cells were examined by flow-cytometric analysis. The mean transduction efficiency was 80% for HLE B-3 cells. Downregulation of c-myc mRNA and protein expression was noticed at 48, 96 and 144 h after the transduction with Ad-AS-myc. Cytostatic effects of Ad-AS-myc in HLE B-3 cells were obvious within 96 h after the transduction. An increased incidence of apoptosis and G1-phase arrest was identified in the Ad-AS-myc-transduced HLE B-3 cells. HLE B-3 cells were successfully transduced with adenovirus-mediated antisense c-myc construct. Ad-AS-myc transduction could significantly inhibit cell proliferation and induce cell apoptosis and G1-phase arrest in HLE B-3 cells. It may provide a novel approach for prevention of posterior capsular opacification.

Interleukin-4 gene transfer into rat pancreas by recombinant adenovirus

Objective. Adenovirus-mediated gene transfer technology may provide a novel approach in the treatment of pancreatic diseases. In the rat model of chronic pancreatitis induced by dibutyltin dichloride (DBTC), Th1 lymphocytes are known to be involved in the mediation of inflammation. We therefore investigated whether local expression of the Th2 cytokine interleukin (IL)-4 might modulate the inflammatory response. To address this question, we have established a protocol of efficient gene transfer into rat pancreas. Material and methods. Recombinant adenovirus constructs carrying the Escherichia coli β-galactosidase gene (Adβ-gal) or the rat IL-4 gene (AdrIL-4) were injected into the left gastric artery of healthy LEW.1W rats. Expression of β-Gal and IL-4 in pancreatic cells was analyzed by X-Gal staining and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. After optimization of the transduction protocol, effects of the IL-4 gene transfer on pancreatic inflammation and fibrosis were studied in DBTC-treated rats. Results. Seven days after Adβ-gal injection, β-gal-positive cells were detectable in the rat pancreas. RT-PCR analysis using RNA from pancreata of AdrIL-4-treated rats indicated that IL-4 was expressed for at least 14 days after adenovirus application. Expression of the IL-4 transgene was accompanied by a transient increase of the IL-10 mRNA level in the pancreas. In DBTC-treated rats, adenovirus-mediated transfer of the IL-4 gene modified the pattern of infiltrating inflammatory cells in the pancreas. Importantly, a decrease of CD4+ helper cells was observed. Conclusions. Our data suggest that the injection of recombinant adenoviruses into the left gastric artery is a promising approach to achieving expression of therapeutic transgenes in the pancreas.

Suppression of gastric cancer growth by adenovirus-mediated transfer of the PTEN gene

To investigate the tumor-suppressive effect of the phosphatase and tensin homologue deleted from chromosome (PTEN) in human gastric cancer cells that were wild type for PTEN. Adenoviruses expressing PTEN or luciferase as a control were introduced into gastric cancer cells. The effect of exogenous PTEN gene on the growth and apoptosis of gastric cancer cells that are wtPTEN were examined in vitro and in vivo. Adenovirus-mediated transfer of PTEN (Ad-PTEN) suppressed cell growth and induced apoptosis significantly in gastric cancer cells (MGC-803, SGC-7901) carrying wtPTEN in comparison with that in normal gastric epithelial cells (GES-1) carrying wtPTEN. This suppression was induced through downregulation of the Akt/PKB pathway, dephosphorylation of focal adhesion kinase and mitogen-activated protein kinase and cell-cycle arrest at the G2/M phase but not at the G1 phase. Furthermore, treatment of human gastric tumor xenografts (MGC-803, SGC-7901) with Ad-PTEN resulted in a significant (P<0.01) suppression of tumor growth. These results indicate a significant tumor-suppressive effect of Ad-PTEN against human gastric cancer cells. Thus, Ad-PTEN may be used as a potential therapeutic strategy for treatment of gastric cancers.

Ciliary neurotrophic factor protects retinal ganglion cells from axotomy-induced apoptosis via modulation of retinal gliain vivo

Adenoviral-mediated transfer of ciliary neurotrophic factor (CNTF) to the retina rescued retinal ganglion cells (RGCs) from axotomy-induced apoptosis, presumably via activation of the high affinity CNTF receptor alpha (CNTFRalpha) expressed on RGCs. CNTF can also activate astrocytes, via its low affinity leukemia inhibitory receptor beta expressed on mature astrocytes, suggesting that CNTF may also protect injured neurons indirectly by modulating glia. Adenoviral-mediated overexpression of CNTF in normal and axotomized rat retinas was examined to determine if it could increase the expression of several glial markers previously demonstrated to have a neuroprotective function in the injured brain and retina. Using Western blotting, the expression of glial fibrillary acid protein (GFAP), glutamate/aspartate transporter-1 (GLAST-1), glutamine synthetase (GS), and connexin 43 (Cx43) was examined 7 days after intravitreal injections of Ad.CNTF or control Ad.LacZ. Compared to controls, intravitreal injection of Ad.CNTF led to significant changes in the expression of CNTFRalpha, pSTAT(3), GFAP, GLAST, GS, and Cx43 in normal and axotomized retinas. Taken together, these results suggest that the neuroprotective effects of CNTF may result from a shift of retinal glia cells to a more neuroprotective phenotype. Moreover, the modulation of astrocytes may buffer high concentrations of glutamate that have been shown to contribute to the death of RGCs after optic nerve transection.

Method-related effects of adenovirus-mediated LacZ and SERCA1 gene transfer on contractile behavior of cultured failing human cardiomyocytes

Introduction: Adenovirus-mediated gene transfer into cardiomyocytes has emerged as an interesting tool to study functional effects of single proteins. However, the functional consequences of cell isolation, cell culture per se and adenovirus-mediated transfer of the LacZ or SERCA1 gene in failing human cardiomyocytes warrant further investigation. Methods: Primary cell culture was performed without or after adenovirus-mediated gene transfer of LacZ or SERCA1. Functional behavior of myocytes was assessed under basal conditions (field stimulation, 0.5 Hz, 37 °C), and during inotropic stimulation with isoproterenol (ISO; 10 −9–10 −5 M), [Ca 2+] o (1.5–15 mM) or increasing stimulation rates (0.25–2.5 Hz). Results were compared to trabeculae from the same hearts. Results: Freshly isolated myocytes showed full inotropic competence as compared to multicellular preparations. The response to stimulation with ISO and [Ca 2+] o, as well as changes in stimulation rate resulted in a maximal increase in fractional cell shortening (FS) to 215±24% and 291±34%, and a frequency-dependent decline in FS to 46±5% of the basal value, respectively. After 48 h of cell culture, basal FS did not change significantly compared to fresh cells but both time to peak shortening and time to 50% relengthening were prolonged. After culture, the concentration–response curve for ISO was significantly shifted to the left (EC 50 5.16×10 −8 vs. 1.12×10 −8 M, p<0.05). LacZ gene transfer caused efficient β-Gal expression without affecting the inotropic responses to ISO or stimulation rate but impaired the contractile amplitude. SERCA1 gene transfer increased FS by 68% vs. LacZ and accelerated relengthening kinetics (+d L/d t 93±13 vs. 61±8 μm/s, p<0.05 vs. LacZ). Discussion: Contractile responses of isolated human myocytes are comparable to multicellular preparations. The use of primary cell culture and adenovirus infection with CMV-promoter-mediated LacZ expression per se modulates contractile behavior in failing human myocytes. SERCA1 expression markedly improves contractile function. The method-related changes in contractile behavior observed here need to be taken into account in further studies.

Fibroblast Growth Factor-2 Stimulates Interleukin-6 Secretion in Human Pancreatic Periacinar Myofibroblasts

Fibroblast growth factor-2 (FGF-2) plays an important role in the pathophysiology of acute and chronic pancreatitis. In the present study, to evaluate the proinflammatory nature of FGF-2, we investigated the effects of FGF-2 on IL-6 secretion in human pancreatic periacinar myofibroblasts. IL-6 supernatant levels were determined by enzyme-linked immunosorbent assays (ELISA). IL-6 mRNA expression were determined by Northern blots and quantitative PCRs. Activated protein (AP)-1 DNA-binding activities were evaluated by electrophoretic gel mobility shift assays (EMSA). FGF-2 induced IL-6 release in a dose- and time-dependent manner. FGF-2 activity for IL-6 induction was the same as that of IL-17. The combination of FGF-2 and IL-17 exerted additive effects at mRNA and protein levels. FGF-2 induced AP-1 DNA-binding activity, but blockage of AP-1 signaling by adenovirus-mediated transfer of a dominant negative c-Jun gene did not affect FGF-2-induced IL-6 mRNA expression. FGF-2 rapidly induced activation of ERK1/2 and p38 MAP kinases, and specific inhibitors for these enzymes significantly reduced FGF-2-induced IL-6 release. In the pancreas, FGF-2 may not only play a role as a growth factor in tissue injury repair processes but also as an inducer of acute-phase response via stimulation of IL-6 release.

Localization of various secretory phospholipase A 2 enzymes in male reproductive organs

Current evidence suggests the presence of transcripts for several secretory phospholipase A 2 (sPLA 2) enzymes in male genital organs. In this study, we examined by immunohistochemistry the localization of group IIA, IIC, IID, IIE, IIF, V and X sPLA 2s in male genital organs. In sPLA 2-IIA-deficient C57BL/6 mouse testis, sPLA 2-IIC, -IID, -IIE, -IIF, -V and -X were diversely expressed in spermatogenic cells within the seminiferous tubules. Immunoblotting revealed the presence of these sPLA 2s in mouse spermatozoa. In addition, sPLA 2-IIF, -V and -X were localized in the interstitial Leydig cells. The same set of sPLA 2s was detected in a mouse cultured Leydig cell line, and adenovirus-mediated transfer of these sPLA 2s into Leydig cells resulted in increased prostaglandin production. sPLA 2-IIC, -IID, -IIE, -IIF, -V and -X were also detected diversely in the epithelium of the epididymis, vas deferens, seminal vesicles, and prostate. In a sPLA 2-IIA-positive FVB strain, weak expression of sPLA 2-IIA was detected in Leydig cells. Notable differences in the sPLA 2 expression profiles were found in the seminal vesicles and prostate between mice and humans. Taken together, individual sPLA 2s exhibit distinct or partially overlapping localizations in male reproductive organs, suggesting both specific and redundant functions.

Effect of Adenoviral-Mediated Transfer of Transforming Growth Factor-β1 on Colonic Anastomotic Healing

Transforming growth factor-beta1 plays a central role in colonic repair. We examined the temporal effect of vector-mediated transfer of transforming growth factor-beta1 on colonic anastomotic healing. Male Sprague-Dawley rats (n = 24) underwent transection of the distal colon and single-layer anastomosis. Proximal to the anastomosis, the colon was again transected and a colostomy was matured proximally. The distal colon was intubated with a silicone catheter, tunneled along subcutaneous tissues, and connected to a swivel apparatus for postoperative luminal infusion. Rats were randomized into four groups (n = 6 each). Two control groups received 10(10) plaque-forming units of a Type 5 E1-deleted adenovirus carrying the bacterial beta-galactosidase gene either immediately following surgery or on postoperative Day 3. The treatment groups received transforming growth factor-beta1 with the same viral construct at parallel time points. On postoperative Day 6, anastomotic bursting pressure and site were determined in situ with the anastomotic tissue subsequently harvested and analyzed by enzyme-linked immunosorbent assay for beta-galactosidase and transforming growth factor-beta1. When compared with its corresponding control, the group that received the transforming growth factor-beta1 gene on postoperative day 3 had a significantly higher bursting pressure (mmHg; 119 +/- 16 vs. 160 +/- 12, mean +/- SD; P = 0.001). While the majority of colons (5/6) from the control group burst at the anastomosis, none of the colons in the group that received transforming growth factor-beta1 on day 3 burst at the anastomotic site (P = 0.007). Beta-Galactosidase levels (pg/ml) in anastomotic tissue were significantly increased in both control groups when compared with their respective treatment groups (101 +/- 43 vs. 38 +/- 30, P = 0.01 when infused the day of surgery and 243 +/- 92 vs. 50 +/- 30, P = 0.009 when infused on day 3). Anastomotic levels of transforming growth factor-beta1 were also increased in the group receiving the transforming growth factor-beta1 gene on day 3 (214 +/- 66 vs. 135 +/- 24, P = 0.02). Gene transfer into the healing colonic anastomosis can be effectively achieved via intraluminal administration of adenoviral vectors. Transfer of transforming growth factor-beta1 increased the strength of colonic anastomoses when given at Day 3 but not at Day 0, demonstrating its diverse effects in the wound healing sequence. Thus, gene transfer of transforming growth factor-beta1 may avoid the need for a diverting stoma in cases of rectal surgery and impaired healing resulting from chemotherapy or radiation.

Identification of SCN3B as a novel p53-inducible proapoptotic gene.

Tumor suppressor p53 is a transcription factor that induces growth arrest and/or apoptosis in response to cellular stress. To identify novel p53-inducible genes, we compared the expression of genes in normal mouse embryo fibroblasts (MEFs) to p53-null cells by cDNA representational difference analysis. We report here that expression of endogenous sodium channel subunit beta 3 (SCN3B) is upregulated in mouse embryonic fibroblasts by DNA damage in a p53-dependent manner. In addition, we found that SCN3B levels are upregulated in human cancer cell lines by DNA damaging agents, as well as by overexpression of p53, but not significantly by p63 or p73. Furthermore, we identified two putative p53-binding sites upstream of the first exon (RE1) and in the third intron (RE2). The p53 protein can directly interact with the putative p53-binding sites in vivo, as assessed by chromatin immunoprecipitation. A reporter gene assay revealed that these two p53-binding sites are functional response elements. The SCN3B protein appears to be localized to the endoplasmic reticulum (ER). Introduction of the SCN3B gene into T98G and Saos2 cells potently suppressed colony formation. Furthermore, we found that adenovirus-mediated transfer of SCN3B induced apoptosis when combined with anticancer agents. The results presented here suggest that SCN3B mediates a p53-dependent apoptotic pathway and may be a candidate for gene therapy combined with anticancer drugs.

Adenoviral mediated transfer of TIMP-3 partially prevents glutamate-induced cell death in primary cultured cortical neurons of the rat

Metalloproteinases and tissue inhibitor of metalloproteinases (TIMPs) are associated with tissue reorganization after injury. The up-regulation of TIMP-3 has recently been found in ischemic brain although its functional implications are unclear. In this study we show that overexpression of TIMP-3 by adenovirus-mediated gene transfer partially protects neurons against excitotoxic death induced by glutamate in culture. The partial neuroprotection afforded by TIMP-3 has implications for our understanding of the physiological role of TIMP-3 in the normal and damaged central nervous system.

Adenoviral-mediated transfer of p53 gene enhances TRAIL-induced apoptosis in human hepatocellular carcinoma cells

p53 is a tumor suppressor protein with numerous biological functions including transformation, regulation of cell growth, differentiation and apoptosis. The TNF-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in various transformed cell lines. We investigated the effects of combining wild-type p53 gene transduction by adenoviral infection (Ad-p53) with addition of TRAIL on cell death, expression levels of TRAIL receptors (TRAIL-R1, TRAIL-R2), FLICE inhibitory protein (FLIP) and X-linked inhibitor of apoptosis protein (XIAP) on human hepatocellular carcinoma (HCC) cell lines. HCC cell death was increased by combination of Ad-p53 infection and addition of TRAIL compared to either alone. Western blotting demonstrated decreased TRAIL-R1 and TRAIL-R2 levels after infection with Ad-p53. FLIP levels decreased in Huh7 cells and Hep3B cells, and XIAP levels decreased in all three HCC cell lines after infection with Ad-p53. Thus, death of HCC cells due to combined p53 gene transduction and exogenous TRAIL may be due to down regulation of FLIP or XIAP.

Adenovirus-Mediated Transfer of Thyroid Transcription Factor-1 Induces Radioiodide Organification and Retention in Thyroid Cancer Cells

Loss of thyroid-specific gene expression and functions accompanied by loss of thyroid transcription factors render them unresponsive to radioiodide therapy in poorly differentiated and anaplastic thyroid cancer. In anticipation of reactivation of thyroid functions, we investigated the effect of thyroid transcription factor-1 (TTF-1) gene transfer on thyroid cancer cells. Reexpression of thyroperoxidase (TPO) and thyroglobulin (Tg) mRNA and protein was detected in poorly differentiated human thyroid cancer cells that were infected with an adenovirus vector containing TTF-1 (AdTTF-1). Although TTF-1 gene transfer faintly induced iodide uptake, the induction of sodium/iodide symporter (NIS) mRNA was not observed in AdTTF-1-infected cells. To analyze the effect of TTF-1 on iodide metabolism, we transfected an NIS expression vector into BHP18-21v cells and cloned a cell line (N-BHP18-21v) that stably expressed NIS. The treatment of N-BHP18-21v cells with AdTTF-1 significantly increased the amount of protein-bound radioiodide and prolonged the iodide efflux. AdTTF-1 injections significantly induced iodide retention and organification in tumors formed from N-BHP18-21v cells in nude mice. These results indicate that AdTTF-1 specifically induces iodide organification and retards iodide efflux in thyroid cancer cells in vitro and in vivo.

Adenovirus-Mediated Transfer of siRNA against Survivin Induced Apoptosis and Attenuated Tumor Cell Growth in Vitro and in Vivo

Gene targeting using short interfering RNA (siRNA) has become a common strategy to explore gene function because of its prominent efficacy and specificity. For the application of siRNA technology to gene therapy, however, still more efficient transduction of siRNA into target cells is needed. In this study, we developed an adenoviral vector harboring a tandem-type siRNA expression unit, in which sense and antisense strands composing the siRNA duplex were separately transcribed by two human U6 promoters. Targeting survivin, an antiapoptotic molecule widely overexpressed in malignancies but not detected in terminally differentiated adult tissues, this type of adenoviral vector (Adv-siSurv) successfully exerted a gene knockdown effect and induced apoptosis in HeLa, U251, and MCF-7 cells. These cancer cells, once infected with Adv-siSurv, displayed remarkably attenuated growth potential, both in vitro and in vivo. Moreover, intratumoral injection of Adv-siSurv significantly suppressed tumor growth in a xenograft model using U251 glioma cells. This novel modality may be a promising tool for cancer therapy.