RESUMO
The expanding use of adenoviral vectors for gene therapy has brought about the need for new analytical tools. We have developed an anion-exchange high-performance liquid chromatography method to analyze recombinant adenovirus serotype 5 samples. Before this assay, available analytical methods consisted of either long-term biological assays or required highly purified test articles. These methods were inadequate for optimizing adenovirus production and purification. This assay can quantitate viral particles in either crude lysates or highly pure samples. It can be used to assess particles in both dilute and concentrated samples over a wide dynamic range. Moreover, the population of viral particles eluted in the peak contains most of the infectious virions. This assay is a sensitive technique that overcomes the limitations of previous methods. It provides an essential tool to accomplish process optimization.
Assuntos
Adenoviridae/isolamento & purificação , Adenoviridae/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , DNA Viral/isolamento & purificação , Vírus Defeituosos/isolamento & purificação , Vírus Defeituosos/metabolismo , Adenoviridae/genética , Western Blotting , Linhagem Celular , Vírus Defeituosos/genética , Eletroforese em Gel de Poliacrilamida , Estudos de Avaliação como Assunto , Vetores Genéticos , UltracentrifugaçãoRESUMO
We have investigated the use of column chromatography for the purification of ACN53, a recombinant adenovirus type 5 encoding the human p53 tumor suppressor protein. Anion exchange, size exclusion, hydrophobic interaction, and metal chelating resins were tested; each was found to have distinct advantages and disadvantages. Based on these data, a rapid method was devised for the purification of ACN53. The resultant product was characterized and compared to cesium chloride density-gradient purified virus by SDS-PAGE, Western blot analysis, absorbance spectrum, total particle-to-infectious particle ratio, expression of p53 gene product in Saos-2 cells, growth inhibition of Saos-2 cells, and contamination by ATCC-293 host cell proteins. The results show that column chromatography offers an alternative to ultracentrifugation for the purification of recombinant adenoviruses for use in human gene therapy trials and other research applications.
Assuntos
Adenovírus Humanos/isolamento & purificação , Cromatografia/métodos , Vetores Genéticos/isolamento & purificação , Proteína Supressora de Tumor p53/genética , Adenovírus Humanos/genética , Resinas de Troca Aniônica/química , Fracionamento Celular , Linhagem Celular , Césio/química , Cloretos/química , Cromatografia de Afinidade/métodos , Cromatografia em Gel/métodos , Endonucleases/metabolismo , Etanolaminas/química , Vetores Genéticos/genética , Humanos , Zinco/químicaRESUMO
Reconstitution of retinoblastoma gene (RB) deficient tumor cells with RB generally leads to growth suppression in vitro and/or reduced tumorigenicity in nude mice. An alternate approach to gene replacement is the delivery of the RB gene product (p110RB) into cells lacking its expression. In this report we demonstrate that exogenously added p110RB is taken up by and localized to the nucleus of cultured cells and has growth suppression properties similar to endogenous RB. RB-negative (RBneg) tumor cells are preferentially growth inhibited while most RB-positive (RBpos) tumor cells and normal cells are much less sensitive. We have extended these studies to relevant nude mouse xenograft models for human lung cancer. Local or systemic administration of p110RB inhibits tumor growth in treated animals. These results represent the first use of a tumor suppressor protein as a potential cancer therapeutic.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Divisão Celular/efeitos dos fármacos , Neoplasias Experimentais/patologia , Proteína do Retinoblastoma/farmacologia , Animais , Núcleo Celular/metabolismo , Inibidores do Crescimento , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Transplante de Neoplasias , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
The role of intra- and extravesicular ascorbate has been investigated in dopamine beta-monooxygenase (D beta M) turnover using adrenal medulla chromaffin granule ghosts. Resealing of vesicle ghosts with high levels of intravesicular ascorbate leads to viable vesicles, as evidenced from the high rates of the ATP-dependent accumulation of tyramine, Vmax = 14 +/- 1 nmol/min.mg and Km = 20 +/- 6 microM. However, the D beta M-catalyzed conversion of tyramine to octopamine occurs slowly, Vmax = 0.50 +/- 0.13 nmol/min.mg and Km = 29 +/- 18 mM. When ascorbate is present instead in the external buffer, the D beta M rate increases 3.6-fold for a final Vmax = 1.8 +/- 0.2 and Km = 1.2 +/- 0.3 mM. This relatively high rate of enzyme turnover is retained in ghosts resealed with a large excess of ascorbate oxidase, ruling out contamination by intravesicular ascorbate as the source of enzyme activity. The synergistic effect of intravesicular ascorbate was examined under conditions of 2 mM external ascorbate, showing that the enzymatic rate increases 2.7-fold, from 1.2 (0 internal ascorbate) to 3.2 +/- 0.4 nmol/min.mg (saturating internal ascorbate). This result confirms that high levels of internal ascorbate are not damaging to intravesicular D beta M. These studies demonstrate very clearly that external ascorbate is the preferred reductant for the membranous form of D beta M in chromaffin granule ghosts.
