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ABSTRACT: We studied the effects of specific retroviral elements in a first-generation serotype 5 adenoviral (Ad5) vector, AdLTR(2)EF1alpha-hEPO. This vector contains 858 base pair (bp) [251-bp envelope sequence plus 607-bp long-terminal repeat (LTR)] from Moloney murine leukemia virus (MoMLV), upstream of the human elongation factor-1alpha (EF1alpha) promoter and human erythropoietin (hEPO) cDNA, with the LTR sequence downstream of the polyadenylation signal. We compared expression of AdLTR(2)EF1alpha-hEPO with corresponding expressions of two conventional Ad5 vectors, AdEF1alpha-hEPO and AdCMV-hEPO, in vivo in submandibular glands in rats. Both the conventional vectors yielded low serum hEPO levels by day 7, and little change in hematocrits. In contrast, after receiving AdLTR(2)EF1alpha-hEPO, the rats showed elevated hEPO levels and hematocrits for 1-3 months. In vitro studies showed that the integration efficiencies of all the vectors were similar (approximately 10(-3)). Approximately 0.1% of the vector genomes were present 1 year after delivery in the case of each of the three vectors, primarily as intact linear double-strand DNA. The unique results seen with AdLTR(2)EF1alpha-hEPO are partly because of LTR enhancer activity. However, other cis-acting activity, which is not immunomodulatory but nevertheless influences promoter methylation, appears to be involved. A vector such as AdLTR(2)EF1alpha-hEPO may prove useful in clinical applications in which extended, but not "permanent," transgene expression is desirable.
Molecular Therapy 07/2008; 16(6):1089-97. · 6.87 Impact Factor
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ABSTRACT: Radiotherapy is commonly used to treat the majority of patients with head and neck cancers. Salivary glands in the radiation field are dramatically affected by this procedure. The purpose of this study was to examine pharmacokinetic characteristics of the stable nitroxide 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (tempol) with respect to radioprotection of the salivary glands.
To evaluate the effect of different doses and times of administration, the heads of C3H mice were exposed to a single irradiation dose of 15 Gy, with i.p. tempol injection. To analyze other routes of administration, we injected 275 mg/kg tempol by an i.m., i.v., or s.c. route, 10 minutes before irradiation. We also tested whether oral administration of tempol in a topical form (either in a mouthwash or gel) provided any salivary gland protection.
Tempol treatment (137.5 or 275 mg/kg, i.p., 10 minutes before irradiation) significantly reduced irradiation-induced salivary hypofunction (approximately 50-60%). I.v. or s.c. administration of tempol also showed significant radioprotection, whereas i.m. administration proved to be ineffective. Topical use of tempol, either as a mouthwash or gel, also was radioprotective.
Our results strongly suggest that tempol is a promising candidate for clinical application to protect salivary glands in patients undergoing radiotherapy for head and neck cancers.
Clinical Cancer Research 11/2005; 11(20):7564-8. · 7.74 Impact Factor
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ABSTRACT: Molecular Therapy (2005) 11, S339|[ndash]|S340; doi: 10.1016/j.ymthe.2005.07.420
877. Long-Term Transgene Expression with a Second Generation Hybrid AdenoRetroviral Vector
Changyu Zheng1, Weitan Zhang1, Joseph M. Vitolo1 and Bruce J. Baum11GTTB, NIDCR, NIH, DHHS, Bethesda, MD
Molecular Therapy 04/2005; · 6.87 Impact Factor
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ABSTRACT: Radiotherapy is commonly used to treat a majority of patients with head and neck cancers. The long-term radiation-induced reduction of saliva output significantly contributes to the posttreatment morbidity experienced by these patients. The purpose of this study was to test the ability of the stable-free radical Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), an established radioprotector, to prevent radiation-induced salivary hypofunction in mice.
The heads of C3H mice were exposed to a range of single radiation doses with or without an i.p. injection of 275 mg/kg Tempol 10 min before treatment. Salivary gland output was assessed 8 weeks postirradiation.
Radiation caused a dose-dependent reduction in salivary flow in this model. Tempol treatment alone significantly reduced radiation-induced salivary hypofunction. The combination of Tempol with mouth/nose shielding showed essentially complete radiation protection at 15 Gy and approximately 75% protection at 17.5 Gy.
This study demonstrates for the first time that significant radioprotection of the salivary glands is possible with Tempol in C3H mice.
Clinical Cancer Research 04/2004; 10(5):1807-12. · 7.74 Impact Factor
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ABSTRACT: Assembly of a DNA fragment containing a Xenopus borealis somatic-type 5S RNA gene into a nucleosome greatly restricts binding of the 5S gene-specific transcription factor IIIA (TFIIIA) to the 5S internal promoter. However, TFIIIA binds with high affinity to 5S nucleosomes lacking the N-terminal tail domains of the core histones or to nucleosomes in which these domains are hyperacetylated. The degree to which tail acetylation or removal improves TFIIIA binding cannot be simply explained by a commensurate change in the general accessibility of nucleosomal DNA. In order to investigate the molecular basis of how TFIIIA binds to the nucleosome and to ascertain if binding involves all nine zinc fingers and/or displacement of histone-DNA interactions, we examined the TFIIIA-nucleosome complex by hydroxyl radical footprinting and site-directed protein-DNA cross-linking. Our data reveal that the first six fingers of TFIIIA bind and displace approximately 20 bp of histone-DNA interactions at the periphery of the nucleosome, while binding of fingers 7 to 9 appears to overlap with histone-DNA interactions. Molecular modeling based on these results and the crystal structures of a nucleosome core and a TFIIIA-DNA cocomplex yields a precise picture of the ternary complex and a potentially important intermediate in the transition from naïve chromatin structure to productive polymerase III transcription complex.
Molecular and Cellular Biology 02/2004; 24(2):697-707. · 5.53 Impact Factor
