Characterization of integration frequency and insertion sites of adenovirus DNA into mouse liver genomic DNA following intravenous injection.

While generally referred to as "non-integrating" vectors, adenovirus vectors have the potential to integrate into host DNA via random, illegitimate (nonhomologous) recombination. The present study provides a quantitative assessment of the potential integration frequency of adenovirus 5 (Ad5)-based vectors following intravenous injection in mice, a common route of administration in gene therapy applications particularly for transgene expression in liver. We examined the uptake level and persistence in liver of first generation (FG) and helper-dependent (HD) Ad5 vectors containing the mouse leptin transgene.

Lack of effects on fertility and developmental toxicity of a quadrivalent HPV vaccine in Sprague-Dawley rats.

Human papillomavirus (HPV) infection is one of the most common sexually transmitted diseases, with approximately half of the HPV-infected people being adolescents and young adults. A recently developed quadrivalent HPV vaccine, GARDASIL((R)), has been shown to be highly effective in the prevention of a number of HPV-mediated diseases. The objective of the present study was to evaluate the potential effects of the vaccine on female fertility and F1 development, growth, behavior, and reproductive performance.

Tumorigenicity assessments of Per.C6 cells and of an Ad5-vectored HIV-1 vaccine produced on this continuous cell line.

PER.C6, a cell line derived from human embryonic retinal cells transformed with the Adenovirus Type 5 (Ad5) E1A and E1B genes, is used to produce E1-deleted Ad5 vectors such as the MRKAd5 HIV-1 gag vaccine. While whole, live PER.

Adaptation of the WHO guideline for residual DNA in parenteral vaccines produced on continuous cell lines to a limit for oral vaccines.

Although there is a WHO guidance for a limit on residual DNA for parenterally administered vaccines produced on continuous cell lines, there is no corresponding guidance for oral vaccines. To help determine an oral limit, we performed a study of Vero cell DNA uptake in rats, in which the relative uptake and persistence of Vero cell DNA administered orally was compared to its uptake when delivered intramuscularly (IM). The results of this study allowed the generation of an empirically derived IM versus oral factor (10(6)) representing the relative inefficiency of DNA uptake by oral administration.

Ensuring the quality, potency and safety of vaccines during preclinical development.

There is an abundance of vaccines currently in development, with most of them exploring novel mechanisms, adjuvants and/or delivery systems not only for traditional prophylactic use, but also for therapeutic uses. As vaccines are generally administered to healthy individuals, ensuring their quality, potency and safety becomes crucial, especially prior to evaluation in humans. To ensure these key attributes, vaccine developers need to incorporate them as early in the development program as possible, starting in basic research and continuing through preclinical, clinical and postmarketing development.

Detection of integration of plasmid DNA into host genomic DNA following intramuscular injection and electroporation.

Plasmid vectors have been widely used for DNA vaccines and gene therapy. Following intramuscular injection, the plasmid that persists is extrachromosomal and integration into host DNA, if it occurs at all, is negligible. However, new technologies for improving DNA delivery could increase the frequency of integration.

Peroxisome proliferators activate growth regulatory pathways largely via peroxisome proliferator-activated receptor alpha-independent mechanisms.

Peroxisome proliferators (PPs) induce liver tumors in rodents through an unknown mechanism requiring PP-activated receptor (PPAR) alpha. Since PPs possess growth modulatory activities that may be important to their hepatocarcinogenicity, we aimed at dissociating the activation of growth signaling pathways from the PPARalpha-mediated response induced by PPs in cultured rat primary hepatocytes. Pretreatment with the differentiation-promoting agent dimethylsulfoxide (DMSO) increased PPARalpha mRNA/protein and enhanced the expression of PPARalpha-regulated genes [fatty acyl Co-A oxidase (FACO), cytochrome P450 4A1 (CYP4A1)] induced by PPs.

Plasmid DNA vaccines: assay for integration into host genomic DNA.

The primary safety concern for DNA vaccines is their potential to integrate into host cellular DNA. We describe a sensitive and quantitative assay for investigating the tissue distribution and integration of plasmid DNA vaccines. By including gonadal tissues in the analysis, the potential for germline transmission is also assessed.

Peroxisome proliferators and fatty acids negatively regulate liver X receptor-mediated activity and sterol biosynthesis.

Peroxisome proliferators (PPs) are potent tumor promoters in rodents. The mechanism of hepatocarcinogenesis requires the nuclear receptor peroxisome proliferator activated receptor-alpha (PPARalpha), but might also involve the PPARalpha independent alteration of signaling pathways that regulate cell growth. Here, we studied the effects of PPs on the mevalonate pathway, a critical pathway that controls cell proliferation.

Plasmid DNA vaccines: tissue distribution and effects of DNA sequence, adjuvants and delivery method on integration into host DNA.

A variety of factors could affect the frequency of integration of plasmid DNA vaccines into host cellular DNA, including DNA sequences within the plasmid, the expressed gene product (antigen), the formulation, delivery method, route of administration, and the type of cells exposed to the plasmid. In this report, we examined the tissue distribution and potential integration of plasmid DNA vaccines following intramuscular administration in mice and guinea pigs. We compared needle versus Biojector (needleless jet) delivery, examined the effect of aluminum phosphate adjuvants, compared the results of different plasmid DNA vaccines, and tested a gene (the human papilloma virus E7 gene) whose protein product is known to increase integration frequency in vitro.

Plasmid DNA vaccines: investigation of integration into host cellular DNA following intramuscular injection in mice.

The primary safety concern for DNA vaccines is their potential to integrate into the host cell genome. We describe an integration assay based on purification of high-molecular-weight genomic DNA away from free plasmid using gel electrophoresis, such that the genomic DNA can then be assayed for integrated plasmid using a sensitive PCR method. The assay sensitivity was approximately 1 plasmid copy/microg DNA (representing approximately 150,000 diploid cells).

Differentiation of rat oval cells after activation of peroxisome proliferator-activated receptor alpha43.

Peroxisome proliferators (PPs) act as nongenotoxic tumor promoters in rodents. Their hepatocarcinogenicity requires the presence of the PP-activated receptor alpha (PPARalpha); however, the exact role played by this transcription factor in the liver, more precisely in liver cell growth and differentiation, is not known. The aim of this study was to investigate the role of PPARalpha in oval cells, which are considered to be closely related to liver stem cells, act as bipotential progenitors for the two main hepatic lineages, and have been implicated as playing a role in several models of liver carcinogenesis.

Peroxisome proliferators activate extracellular signal-regulated kinases in immortalized mouse liver cells.

Peroxisome proliferators (PPs) are a class of nongenotoxic carcinogens in the rodent liver. The induction of immediate-early gene expression in immortalized mouse liver cells by the PPs Wy-14, 643, monoethylhexyl phthalate, ciprofibrate ethyl ester, and clofibrate suggested that they may be activating growth-regulatory signal transduction pathways. We report that incubation of quiescent ML457 cells with Wy-14,643 resulted in the appearance of two tyrosine-phosphorylated bands of approximately 44 and 42 kDa with maximal phosphorylation at 20 min.

Induction of cyclooxygenase-2 expression by peroxisome proliferators and non-tetradecanoylphorbol 12,13-myristate-type tumor promoters in immortalized mouse liver cells.

Increased expression of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostaglandin synthesis, has been associated with growth regulation and carcinogenesis in several systems. COX-2 is known to be induced by cytokines and the skin tumor promoter 12-tetradecanoylphorbol-13-myristate (TPA). In the present study, we investigated the effects of several non-TPA-type tumor promoters on COX-2 expression in immortalized mouse liver cells.

Growth regulation by peroxisome proliferators: opposing activities in early and late G1.

Peroxisome proliferators (PPs) are a diverse group of nongenotoxic rodent liver carcinogens. One potential mechanism for the carcinogenicity of PPs is epigenetic modulation of growth-regulatory signal transduction pathways. We investigated the effects of PPs on growth-regulatory gene expression and cell proliferation in immortalized mouse liver cells, comparing PPs with other growth regulators and tumor promoters of known activity.

Potential DNA vaccine integration into host cell genome.

Studies have been designed to examine the potential integration of DNA vaccines into the host cell genome. This is of concern because of the possibility of insertional mutagenesis resulting in the inactivation of tumor suppressor genes or the activation of oncogenes. The requirements for adequate testing were determined to be (1) a method to purify host cell genomic DNA from nonintegrated free plasmid, (2) a sensitive method to detect integrated plasmid in the purified genomic DNA, and (3) stringent methods to avoid contamination.

Induction of minisatellite DNA rearrangements by genotoxic carcinogens in mouse liver tumors.

We investigated whether somatic rearrangements in minisatellite DNA are more frequent in chemically induced mouse liver tumors than they are in spontaneous tumors. CD-1 mouse liver tumors were induced by either a single dose or 15 consecutive daily doses of 7,12-dimethylbenz[alpha]anthracene, 4-aminoazobenzene, N-hydroxy-2-acetyl-aminofluorene or diethylnitrosoamine (DEN). Using DNA fingerprinting analysis, we found that the single- and multiple-dose carcinogen treatments caused a 2- to 5-fold higher frequency of minisatellite DNA rearrangements compared with that found in spontaneous tumors--with the exception of single-dose DEN tumors, which showed no increase in rearrangements.

Dose-related changes in the profile of ras mutations in chemically induced CD-1 mouse liver tumors.

We investigated the role of dosing regimen on ras mutations in chemically induced CD-1 mouse liver tumors. The spectra of ras gene mutations in liver tumors that were induced by 15 daily i.p.

Minisatellite DNA-binding proteins in mouse brain, liver, and kidney.

Minisatellites are tandemly repeated DNA sequences that are found in most higher eukaryotes. They are genetically unstable and often gain or lose repeat units. Minisatellite repeats contain a "core" sequence which is highly conserved among a family of minisatellites.

Measurement of plasma angiotensin II: purification by cation-exchange chromatography.

Measurement of plasma angiotensin II (AII) by radioimmunoassay (RIA) usually requires prior purification of the plasma to remove substances that cross-react in the RIA, most notably angiotensin III (AIII). Purification of AII is generally accomplished by solid-phase extraction (SPE) followed by reverse-phase HPLC, with tedious evaporation and resuspension steps in between, and requires collection of many HPLC fractions per sample for RIA. In this report, we describe a rapid two-step SPE procedure for the purification of plasma AII, including an improved protease inhibitor cocktail for preventing the formation or degradation of AII in vitro.

Activation of immediate-early gene expression by peroxisome proliferators in vitro.

The hepatocarcinogenicity of peroxisome proliferators (PPs) in rodents has been attributed both to oxidative DNA damage resulting from excessive leakage of peroxisomal H2O2 and to increased hepatocellular replication that may be independent of peroxisome proliferation. Because of the growing association between tumor promotion and alterations in growth-regulatory signal transduction pathways, we investigated whether PPs can modulate these pathways in a mouse liver epithelial cell line, BNL-CL.2.

Reversal of transformed phenotypes by antisense fos.

The therapeutic use of antisense DNA has started a revolution in pharmacology. As a model system for demonstrating the therapeutic power of the antisense concept, we sought to interrupt signal transduction in H-ras transformed cells to attempt to down-regulate their oncogenic phenotype. We hypothesized that down-regulation of c-fos translation by antisense-fos expression would decrease oncogenic signal transduction through the fos pathway and thus reverse the tumorigenic phenotype of these cells.

Activation of the Ha-, Ki-, and N-ras genes in chemically induced liver tumors from CD-1 mice.

We compared the profile of ras gene mutations in spontaneous CD-1 mouse liver tumors with that found in liver tumors that were induced by a single i.p. injection of either 7,12-dimethylbenz(a)anthracene (DMBA), 4-aminoazobenzene, N-hydroxy-2-acetylaminofluorene, or N-nitrosodiethylamine.