A facile synthesis of [N1,NH(2-)(15)N(2)], [N3,NH(2-)(15)N(2)] and [N2, N3,NH(2-)(15)N(3)]-labeled adenine.
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Adenine↗
Biomedical subjects
Y P Tsao
Publications and source records attributed to Y P Tsao.
The solution structure of [d(CGC)r(amamam)d(TTTGCG)]2.
The solution structure and hydration of a DNA.RNA hybrid chimeric duplex [d(CGC)r(amamam)d(TTTGCG)]2 in which the RNA adenines were substituted by 2'-O-methylated riboadenines was determined using two-dimensional NMR, simulated annealing, and restrained molecular dynamics. Only DNA residue 7T in the 2'-OMe-RNA.DNA junction adopted an O4'-endo sugar conformation, while the other DNA residues including 3C in the DNA.2'-OMe-RNA junction, adopted C1'-exo or C2'-endo conformations. The observed NOE intensity of 2'-O-methyl group to H1' proton of 4am at the DNA.2'-OMe-RNA junction is much weaker than those of 5am and 6am. The 2'-O-methyl group of 4am was found to orient towards the minor groove in the trans domain while the 2'-O-methyl groups of 5am and 6am were found to be in the gauche (+) domain. In contrast to the long-lived water molecules found close to the RNA adenine H2 and H1' protons and the methyl group of 7T in the RNA-DNA junction of [d(CGC)r(aaa)d(TTTGCG)]2, there were no long-lived water molecules found in [d(CGC)r(amamam)d(TTTGCG)]2. This is probably due to the hydrophobic enviroment created by the 2'-O-methylated riboadenines in the minor groove or due to the wider minor groove width in the middle of the structure. In addition, the 2'-O-methylation of riboadenines in pure chimeric duplex increses its melting temperature from 48.5 degrees C to 51.9 degrees C. The characteristic structural features and hydration patterns of this chimeric duplex provide a molecular basis for further therapeutic applications of DNA.RNA hybrid and chimeric duplexes with 2'-modified RNA residues.
Base Sequence↗
Suppression of choroidal neovascularization by adeno-associated virus vector expressing angiostatin.
PURPOSE: To test the efficacy of a recombinant adeno-associated virus (rAAV) vector that expresses mouse angiostatin in suppressing experimental choroidal neovascularization (CNV) in a rat model. METHODS: An rAAV vector, rAAV-angiostatin, was constructed to deliver the mouse angiostatin gene. rAAV-angiostatin and a control virus, rAAV-lacZ, were delivered in vivo by subretinal injection in Brown Norway rats, and the delivery was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). For a CNV suppression experiment, CNV was generated by fundus krypton laser photocoagulation 7 days after the viral vector injection and was evaluated by fluorescein angiography (FA) and histology. Apoptosis in retina was analyzed using the TUNEL assay. Inflammation in the retina was investigated by immunohistochemistry, using antibodies that recognize lymphocytes. RESULTS: rAAV-angiostatin injection led to sustained expression of the angiostatin gene in chorioretinal tissue for up to150 days. FA analysis revealed significant reduction of the average sizes of CNV lesions in rAAV-angiostatin-injected eyes when compared with rAAV-lacZ-injected eyes at both 14 (P = 0.019) and 150 (P = 0.010) days after injection. Moreover, histologic analysis of CNV lesions also revealed significantly smaller lesions in rAAV-angiostatin-injected eyes (P = 0.004). As for adverse effects, rAAV-angiostatin injection did not cause inflammation or apoptosis of cells in retina and choroid. CONCLUSIONS: This is the first report that subretinal injection of rAAV-angiostatin can significantly reduce the sizes of CNV lesions. This and the absence of apoptosis and inflammation in chorioretinal tissue indicate the feasibility of a gene therapy approach for treatment of CNV disease.
Angiostatins↗
Endothelial cell loss induced by phacoemulsification occurs through apoptosis.
BACKGROUND: Cornea endothelial cells are nondividing cells containing pumping function which is crucial for cornea clarity and integrity. Endothelial cell loss occurs after cataract surgical procedures such as phacoemulsification. The authors hypothesize that endothelium damage occurs through apoptosis. METHODS: Ultrasound was achieved by placing a phacoemulsification probe in the anterior chamber and delivering 0% or 50% of maximum power for 2.5 min. The corneal tissue was harvested immediately, and at 1 and 7 days after the operation. Corneal tissue was stained by hemotoxylin and eosin (H&E) and evaluated by light microscopy. Endothelium apoptosis was monitored using the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assay to detect DNA fragmentation. RESULTS: In corneas which underwent phacoemulsification with 0% energy, no cell loss or apoptosis was identified immediately, 1 or 7 days after the operation. Likewise, in corneas exposed to 50% energy, no cell loss or apoptosis was detected immediately after phacoemulsification. However, minimal amount of cell loss but prominent apoptosis was detected with the TUNEL assay 1 day after the operation, whereas significant cell loss but no apoptosis was detected 7 days after the operation by H&E stain. CONCLUSIONS: These results demonstrate that corneal endothelial cell loss induced by ultrasound damage occurs through apoptosis.
Animals↗
Bcl-2 accelerates retinoic acid-induced growth arrest and recovery in human gastric cancer cells.
The role of Bcl-2 as an anti-apoptotic protein has been well documented. In the present work, we present evidence that Bcl-2 may also be involved in cell growth regulation. SC-M1 is an unique cell line which responds to retinoic acid (RA) treatment with reversible growth arrest [Shyu, Jiang, Huang, Chang, Wu, Roffler and Yeh (1995) Eur. J. Cancer 31, 237-243]. In this study, when treated with RA, SC-M1/Bcl2 cells, which were generated by transfecting SC-M1 cells with bcl-2 DNA, were growth-arrested two days earlier than SC-M1/neo cells, which were generated by transfecting SC-M1 cells with vector DNA. This indicates that Bcl-2 accelerates RA-induced growth arrest. In addition to the accelerated growth arrest, RA-treated SC-M1/Bcl2 cells also recovered from growth arrest two days faster than SC-M1/neo cells after the removal of RA. Previously, we had identified the cyclin-dependent kinase inhibitor p21((WAF1/CIP1)) (p21) as a mediator of RA-induced growth arrest [Tsao, Li, Kuo, Liu and Chen (1996) Biochem. J. 317, 707-711]. In a search for the mechanism by which Bcl-2 affects growth regulation, we found that p21 gene expression was more prominent in SC-M1/Bcl2 cells than in SC-M1/neo cells in the presence of RA, but when RA was removed, p21 gene expression levels in SC-M1/Bcl2 cells were also reduced earlier than in SC-M1/neo cells. The present report is the first to show that Bcl-2 accelerates not only growth arrest but also recovery from growth arrest. Moreover, the close correlation between the effect of Bcl-2 on both RA-induced growth arrest and RA-induced p21 gene expression suggests the possibility that Bcl-2 affects cell growth through the mechanism of p21.
Cell Cycle↗
Transcriptional repression of p21((Waf1/Cip1/Sdi1)) gene by c-jun through Sp1 site.
Previously, we found that c-jun represses the tumor suppressor p21((Waf1/Cip1/Sdi1)) (p21) gene expression. In this study, we further investigated the mechanism of the inhibitory effect of c-jun on p21. After analysis of a series of deletion and point mutants of p21 promoter, we found that Sp1-3 site (-77 and -83) relative to the transcription start site played an important role for c-jun-repressing-responsive element in the p21 promoter. Both Sp1 and Sp3 transcription factors were the key factors for this event. However, the data from electrophoretic mobility shift assay indicated that c-jun did not change the Sp1 DNA-binding affinity, suggesting that additional factors may be involved in the repression of p21 by c-jun. Furthermore, c-jun could inhibit butyrate-inducing p21 gene expression through Sp1, indicating at least one common pathway whereby p21 expression is affected by c-jun and butyrate in opposing actions. Moreover, the hyperphosphorylated retinoblastoma protein (Rb) increased in c-jun expressing cells, indicating that phosphorylated Rb may play a role in regulating Sp1 to repress p21 expression. This is the first demonstration of how housekeeping factors and oncogene product counteract the function of tumor suppressor genes to control cell cycle progression.
Binding Sites↗
Therapy and prevention of arthritis by recombinant adeno-associated virus vector with delivery of interleukin-1 receptor antagonist.
OBJECTIVE: To evaluate the recombinant adeno-associated virus vector encoding interleukin-1 receptor antagonist (rAAV-IL-1Ra) complementary DNA for its potential in the treatment and prevention of lipopolysaccharide (LPS)-induced arthritis. METHODS: The therapeutic effect of rAAV-IL-1Ra on arthritis was studied by injecting knees of Sprague-Dawley rats with LPS and rAAV-IL-1Ra and then evaluating the severity of arthritis by leukocyte counts in synovial fluid, histologic changes of synovium, and uptake of 67Ga citrate in joint tissue. To study the therapeutic effect on recurrent arthritis, we induced recurrent arthritis by a second injection of LPS 80 days after primary LPS and rAAV-IL-1Ra injections and then evaluated the severity of recurrent arthritis. To study the prevention of arthritis, rAAV-IL-1Ra was injected into normal joints. After 100 days, LPS was used to induce arthritis, and the severity of arthritis was evaluated. RESULTS: The production of the rAAV-IL-1Ra transgene was up-regulated by LPS-induced joint inflammation and proved to be efficacious in the therapeutic and preventative protocols. Not only primary but also recurrent arthritis could be suppressed by a single injection of rAAV-IL-1Ra. We found that the transgene expression of IL-1Ra could be reactivated by a second challenge with LPS delayed for 80 days after rAAV administration. The therapeutic level of IL-1Ra protein reached a mean +/- SD of 5.8+/-0.5 ng/ml in synovial fluid. In addition, the rAAV transgene persisted within normal joints for at least 100 days and could still be induced to express, after LPS insult, a high level of IL-1Ra (mean +/- SD 5.2+/-0.8 ng/ml) that prevented the occurrence of arthritis. CONCLUSION: This gene therapy, by combining highly efficient and stable rAAV gene delivery, disease-regulated gene expression, and the antiinflammatory effect of IL-1Ra, provides a valuable approach for long-term protection against, and prevention of, arthritis.
Animals↗
Recombinant adeno-associated virus vector expressing glial cell line-derived neurotrophic factor reduces ischemia-induced damage.
To explore the potential of using the recombinant adeno-associated viral (rAAV) vector, expressing glial cell line-derived neurotrophic factor (GDNF) as the gene therapy for stroke, we injected rAAV vectors expressing GDNF (rAAV-GDNF) into the cortex of rats which had been experiencing transient bilateral common carotid artery ligation and right middle cerebral artery ligation for 90 min. GDNF levels in cortical tissues of rAAV-GDNF-injected animals were significantly higher than in the control animals injected with rAAV-expressing lacZ (rAAV-lacZ), indicating that rAAV can deliver and express the GDNF gene in cortical tissues. Triphenyltetrazolium chloride tissue stain analysis revealed that the rAAV-delivered GDNF gene could rescue the brain tissues from ischemia-induced injury. Cortical tissues which received rAAV-GDNF injections had both significantly smaller total volumes of infarction and smaller areas of infarction on each brain slice than those which were injected with rAAV-lacZ. An in situ labeling analysis demonstrated significantly less apoptotic cells in cortical tissues rescued by rAAV-GDNF, indicating prevention of apoptosis as the mechanism of cortical cell protection. Moreover, immunohistochemistry staining of Neu-N indicated that the rescued brain tissues contained the same number of Neu-N-positive neuronal cells as contralateral undamaged brain tissues. This provides strong evidence that cortical neuronal cells can be rescued by GDNF gene therapy. Indeed, these findings show that the rAAV is a potential delivery vector of GDNF gene for the therapy of stroke.
Adenoviridae↗
Sequence variants and functional analysis of human papillomavirus type 16 E5 gene in clinical specimens.
Previously, we found that the E5 protein can be expressed in HPV-16 infected precancerous lesions and cervical cancer. In this study, we investigated the presence of sequence variants of E5 in HPV-16 infected tissues. Toward this end, we amplified the E5 gene by polymerase chain reaction from 29 HPV-16 infected tissues including eight normal tissues, seven high grade neoplastic tissues (high grade squamous intraepithelial lesions (HSIL) and 14 cervical cancer tissues. Sequence analysis demonstrated that there were three mutational hot spots at positions 3979, 4042, and 4077 of the HPV-16 DNA; these and other mutations resulted in six variants in the E5 sequence. This resulted in four E5 protein mutants, named WTE5 [wild type E5 protein], 14E5, 21E5 and 56E5. Functional analysis of these four mutant proteins revealed that the transforming activities of 14E5, 21E5 and 56E5 were 0.95, 0.59, and 0.89 fold of WTE5, respectively. Although E5 was expressed in all of the HSIL and cervical cancer tissues, but in only one of the eight normal tissues tested, only WT E5 protein was found in HSIL while in cervical cancer tissues both WT and mutant E5 proteins were detected. Since these E5 proteins exhibited the same in vitro transforming activity, these data suggest that expression of E5 is important in development and progression toward malignancy but mutation of E5 does not affect the transformation process.
Base Sequence↗
Induction of CD8 T cells by vaccination with recombinant adenovirus expressing human papillomavirus type 16 E5 gene reduces tumor growth.
The potential of the E5 protein as a tumor vaccine candidate has not been explored yet. In this study, we evaluate the human papillomavirus type 16 (HPV-16) E5 protein delivered by an adenovirus vector as a tumor vaccine for cervical lesions. The results demonstrate that a single intramuscular injection of a recombinant adenovirus carrying the HPV-16 E5 gene into syngeneic animals can reduce the growth of tumors which contain E5 gene expression. Moreover, the E5 vaccine-induced tumor protection occurs through CD8 T cells but not through CD4 T cells in in vitro assays. In addition, our studies using knockout mice with distinct T-cell deficiencies confirm that cytotoxic T-lymphocyte-induced tumor protection is CD8 dependent but CD4 independent. Hence, HPV-16 E5 can be regarded as a tumor rejection antigen.
Adenoviridae↗
Full functional rescue of a complete muscle (TA) in dystrophic hamsters by adeno-associated virus vector-directed gene therapy.
Limb girdle muscular dystrophy (LGMD) 2F is caused by mutations in the delta-sarcoglycan (SG) gene. Previously, we have shown successful application of a recombinant adeno-associated virus (AAV) vector for genetic and biochemical rescue in the Bio14.6 hamster, a homologous animal model for LGMD 2F (J. Li et al., Gene Ther. 6:74-82, 1999). In this report, we show efficient and long-term delta-SG expression accompanied by nearly complete recovery of physiological function deficits after a single-dose AAV vector injection into the tibialis anterior muscle of the dystrophic hamsters. AAV vector treatment led to more than 97% recovery in muscle strength for both the specific twitch force and the specific tetanic force, when compared to the age-matched control. Vector treatment also prevented pathological muscle hypertrophy and resulted in normal muscle weight and size. Finally, vector-treated muscle showed substantial improvement of the histopathology. This is the first report of successful functional rescue of an entire muscle after AAV-mediated gene delivery. This report also demonstrates the feasibility of in vivo gene therapy for LGMD patients by using AAV vectors.
Animals↗
Recombinant adeno-associated virus expressing human papillomavirus type 16 E7 peptide DNA fused with heat shock protein DNA as a potential vaccine for cervical cancer.
In this study, we explore a potential vaccine for human papillomavirus (HPV)-induced tumors, using heat shock protein as an adjuvant, a peptide vaccine for safety, and adeno-associated virus (AAV) as a gene delivery vector. The tumor vaccine was devised by constructing a chimeric gene which contained HPV type 16 E7 cytotoxic T-lymphocyte (CTL) epitope DNA (M. C. Feltkamp, H. L. Smits, M. P. Vierboom, R. P. Minnaar, B. M. de Jongh, J. W. Drijfhout, J. ter Schegget, C. J. Melief, and W. M. Kast, Eur. J. Immunol. 23:2242-2249, 1993) fused with the heat shock protein gene as a tumor vaccine delivered via AAV. Our results demonstrate that this vaccine can eliminate tumor cells in syngeneic animals and induce CD4- and CD8-dependent CTL activity in vitro. Moreover, studies with knockout mice with distinct T-cell deficiencies confirm that CTL-induced tumor protection is CD4 and CD8 dependent. Taken together, the evidence indicates that this chimeric gene delivered by AAV has potential as a cervical cancer vaccine.
Animals↗
Simian virus 40 T antigen induces p53-independent apoptosis but does not suppress erbB2/neu gene expression in immortalized human epithelial cells.
Previously, we have shown that simian virus 40 (SV40) T antigen can directly cause apoptosis in immortalized human epithelial cells under normal growth conditions. In this study, we further characterized the mechanism of T-antigen-mediated apoptosis involving p53 and whether T antigen can suppress erbB2/neu gene expression. Our results show the differential regulation of erbB2/neu gene expression in different cell clones in response to T antigen transgene, indicating that the regression of erbB2/neu gene by SV40 T is cell-type dependent. Our previous study reported T-antigen-induced apoptosis in p53 mutant cells; however, in this study we find increased levels of p53 protein in T-antigen-containing cells. Therefore, we examined the transactivation function of p53 in these cells. Our data show the failure to transactivate p53, suggesting that increased p53 protein in T antigen expressing cells is functionless at least for transcriptional activation.
Antigens, Viral, Tumor↗
The expression of type I growth factor receptors in the squamous neoplastic changes of uterine cervix.
AIM: The type I family of growth factor receptors includes ErbB1, ErbB2, ErbB3, and ErbB4 which are frequently overexpressed in various human cancer cells. In this study, we systematically investigated the frequency and distribution of these four receptors in relation to neoplastic changes and tumor behaviors in the uterine cervix. MATERIALS: A total 84 of cases including 12 cases of normal cervical tissues, 6 cases of low grade squamous intraepithelial lesion, 10 cases of high grade squamous intraepithelial lesion, and 56 cases of squamous cells carcinoma were examined. RESULTS: Our results show significant difference with increasing grades of dysplasia in terms of these four receptor expressions. No association was found between these four receptors and cell keratinization/differentiation of squamous cell carcinoma of the cervix. Of the four receptors studied, only the expression of erbB2/neu gene was significantly associated with lymph nodal metastasis. Moreover, we find that the coexpression of ErbB1 and ErbB4 was significant in cervical carcinoma. CONCLUSIONS: The coexpression of ErbB1 and ErbB4 in cervical carcinoma suggests that they may be involved in receptor heterodimerization leading to the activation of signaling pathway in the cervical carcinoma.
Carcinoma, Squamous Cell↗
rAAV vector-mediated sarcogylcan gene transfer in a hamster model for limb girdle muscular dystrophy.
The limb girdle muscular dystrophies (LGMD) are a genetically and phenotypically heterogeneous group of degenerative neuromuscular diseases. A subset of the genetically recessive forms of LGMD are caused by mutations in the four muscle sarcoglycan genes (alpha, beta, gamma and delta). The coding sequences of all known sarcoglycan genes are smaller than 2 kb, and thus can be readily packaged in recombinant adeno-associated virus (rAAV) vectors. Previously, we have demonstrated highly efficient and sustained transduction in mature muscle tissue of immunocompetent animals with rAAV vectors. In this report, we utilize recombinant AAV containing the delta-sarcoglycan gene for genetic complementation of muscle diseases using a delta-sarcoglycan-deficient hamster model (Bio 14.6). We show efficient delivery and widespread expression of delta-sarcoglycan after a single intramuscular injection. Importantly, rAAV vector containing the human delta-sarcoglycan cDNA restored secondary biochemical deficiencies, with correct localization of other sarcoglycan proteins to the muscle fiber membrane. Interestingly, restoration of alpha-, as well as beta-sarcoglycan was homogeneous and properly localized throughout transduced muscle, and appeared unaffected by dramatic overexpression of delta-sarcoglycan in the cytoplasm of some myofibers. These results support the feasibility of rAAV vector's application to treat LGMD by means of direct in vivo gene transfer.
Animals↗
Disease-inducible transgene expression from a recombinant adeno-associated virus vector in a rat arthritis model.
Rheumatoid arthritis (RA) is a systemic autoimmune disease affecting 1% of the world's population, with significant morbidity and mortality. In this study, we investigated a recombinant adeno-associated virus (rAAV) vector for its potential application in RA gene therapy. rAAV encoding Escherichia coli beta-galactosidase was injected into rat joints which had already been induced into acute arthritis after local lipopolysaccharide (LPS) administration, and the efficiency of in vivo transduction was evaluated. We observed a striking correlation between vector transgene expression and disease severity in arthritic joints. The inflammatory reaction peaked at 3 to 7 days after LPS treatment, and, at the same time, 95% of the synoviocytes had high-level transgene expression. Gene expression diminished to the basal level (5%) when the inflammation subsided at 30 days after LPS treatment. More importantly, the diminished transgene expression could be efficiently reactivated by a repeated insult. The transgene expression in normal joints transduced with rAAV remained low for a long period of time (30 days) but could still be induced to high levels (95%) at 3 to 7 days after LPS treatment. This is the first demonstration of disease state-regulated transgene expression. These findings strongly support the feasibility of therapeutic as well as preventative gene transfer approaches for RA with rAAV vectors containing therapeutic genes, which are expected to respond primarily to the disease state of the target tissue.
Animals↗
Adenovirus-mediated p21((WAF1/SDII/CIP1)) gene transfer induces apoptosis of human cervical cancer cell lines.
p21((WAF1/SDII/CIP1)) (p21) arrests cell growth by inhibiting cyclin-depend kinases. To explore the potential of using p21 for the gene therapy of cervical cancer, we infected human papillomavirus (HPV)-positive cervical cancer cells (HeLa, SiHa, and Z172) and HPV-negative cervical cancer cells (C33A) with recombinant adenovirus encoding p21 cDNA. The results revealed that effective inhibition of cell growth could be achieved by sense p21 adenovirus but not antisense p21 adenovirus infection and occurred through apoptosis as measured by DNA fragmentation and chromatin condensation. Apoptosis was also observed in xenografts of human cervical cancer cells infected with sense p21 adenovirus, as confirmed by in situ terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL). The apoptosis was not prevented by overexpression of the bcl-2 transgene. To sum up, the apoptotic effect suggests that p21 should be a tumoricidal agent instead of a tumoristatic agent in preventing cervical cancers. In addition, our report substantiates the combination of the high efficiency of adenovirus vector-mediated gene delivery and the apoptotic effect of p21.
Adenoviridae↗
Apoptosis is induced in aging SV40 T antigen-transformed human fibroblasts through p53- and p21CIP1/WAF1-independent pathways.
When comparing SV40 T antigen-transformed human fibroblasts of a younger generation (24 population doubling) and aging stage (58 population doubling), we found that detachment of cells from the culture surface occurred more frequently in aging cells. DNA fragmentation and chromatin condensation which are typical findings of apoptosis occurred more frequently in aging cells as compared to cells of a younger generation. There is no increase in the p53 level or decrease in the SV40 T antigen level in aging cells as compared to cells of a younger generation. Retinoic acid treatment which can effectively suppress p21 gene expression did not prevent apoptosis. These findings indicate that apoptosis that occurs due to aging-transformed human fibroblasts is mediated through p53- and p21-independent pathways.
Antigens, Polyomavirus Transforming↗