Adenovirus-mediated expression of green fluorescent protein

Martin, Rainer de; Raidl, Maria; Hofer, Erhard; Binder, Bernd R.

doi:10.1038/sj.gt.3300408

Cited by 61 publications

(45 citation statements)

References 9 publications

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“…1 rAd-GFP is an E1-deleted adenoviral vector encoding GFP. 7 Viruses were grown on A549 cells, purified by HPLC, 8 and concentrations determined in particles/ml upon elution from a Resource Q anion exchange HPLC. 9 …”

Section: Virusesmentioning

confidence: 99%

Inefficient killing of quiescent human epithelial cells by replicating adenoviruses: potential implications for their use as oncolytic agents

Vaillancourt¹,

Atencio²,

Quijano³

et al. 2005

Cancer Gene Ther

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Cultured primary human cells have been widely used to assess the selectivity of oncolytic viruses as potential anticancer agents. As culture conditions can potentially have a significant impact on virus replication and ultimately cell killing, we evaluated the effects of dl309, a wild-type adenovirus, and dl01/07, a conditionally replicating adenovirus mutant, on quiescent and proliferating primary mammary epithelial cells. When primary cells were induced into quiescence, both viruses exhibited similar attenuated cell killing. However, cell killing by dl309 was superior to dl01/07 in proliferating primary cells. Analysis of viral effects at the level of entry, E2F activation, DNA replication, and late gene expression indicated that attenuation of dl309 in quiescent cells correlated with decreased expression of viral late genes such as hexon. In contrast, attenuation of dl01/07 in quiescent cells correlated with inefficient induction of E2F activity and inability to undergo efficient DNA replication. In proliferating cells, dl309 replicated efficiently, whereas dl01/07 still showed attenuated replication. In summary, our results indicate the intrinsic preference of wildtype adenoviruses for killing proliferating cells, which is an attractive feature for using adenoviruses as oncolytic agents. These results also highlight the need for the use of appropriate growth conditions for primary cells in vitro to distinguish subtle differences in cell killing among various oncolytic viruses. Cancer Gene Therapy (2005) R eplication-competent adenoviruses are currently being evaluated as novel anticancer agents. A number of modifications in the genome of the adenovirus have been reported to result in viruses that are capable of selectively replicating in and killing cancer cells. 1,2 Based on their ability to selectively kill tumor cells in cell culture and antitumor activity in xenograft tumor models, a few of these engineered viruses are currently being evaluated as oncolytic agents in the clinic. 3 As human adenoviruses do not replicate well in nonhuman cells, it has not been possible to study toxicity related to replication in nontarget tissues in preclinical animal models. In the absence of suitable in vivo models, analysis of selective replication is widely performed by studying these viruses for their effect in vitro on tumor cells lines and primary nontransformed cells. 4 The effect of oncolytic viruses in vitro has been determined at the level of viral DNA replication, late gene expression, induction of cell death, and production of progeny virion. 5 In the in vitro assays, whether cells are rapidly growing or arrested can potentially have a significant effect on virus replication and ultimately cell killing.In the present study, we sought to compare the effect of the wild-type adenovirus and a mutant that was previously reported to replicate preferentially in tumor cells for their effects in proliferating and quiescent normal cells. Our results show that in vitro, replication competent adenoviruses including wild ...

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Section: Virusesmentioning

confidence: 99%

Inefficient killing of quiescent human epithelial cells by replicating adenoviruses: potential implications for their use as oncolytic agents

Vaillancourt¹,

Atencio²,

Quijano³

et al. 2005

Cancer Gene Ther

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“…Purification of large batches of the recombinant adenoviruses was done by two consecutive cesium chloride centrifugations (28). Adenoviruses without cDNA inserts and viruses expressing GFP (26) were grown in parallel and used as controls.…”

Section: Methodsmentioning

confidence: 99%

“…Recombinant Adenoviral Constructs and Infection-Construction of recombinant adenoviruses was done as previously described (25,26). NAB2 and NAB2.AS cDNAs (16) were first subcloned into the XmaI site of the pBluescript II SK ϩ/Ϫ vector and then transferred to the BamHI site of the vector pACCMVpLpASRϩ (24).…”

Section: Methodsmentioning

confidence: 99%

NAB2, a Corepressor of EGR-1, Inhibits Vascular Endothelial Growth Factor-mediated Gene Induction and Angiogenic Responses of Endothelial Cells

Lucerna¹,

Mechtcheriakova²,

Kadl³

et al. 2003

Journal of Biological Chemistry

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In this study we have investigated the role of a specific corepressor of EGR-1, NAB2, to down-regulate vascular endothelial growth factor (VEGF)-induced gene expression in endothelial cells and to inhibit angiogenesis. Firstly, we show a reciprocal regulation of EGR-1 and NAB2 following VEGF treatment. During the initial phase EGR-1 is rapidly induced and NAB2 levels are down-regulated. This is followed by a reduction of EGR-1 and a concomitant increase of NAB2. Secondly, using the tissue factor gene as a readout for VEGFinduced and EGR-1-regulated gene expression we demonstrate that NAB2 can completely block VEGF-induced tissue factor reporter gene activity. Thirdly, by adenovirus-mediated expression we show that NAB2 inhibits up-regulation of tissue factor, VEGF receptor-1, and urokinase plasminogen activator mRNAs even when a combination of VEGF and bFGF is used for induction. In addition, NAB2 overexpression significantly reduced tubule and sprout formation in two different in vitro angiogenesis assays and largely prevented the invasion of cells and formation of vessel-like structures in the murine Matrigel model. These data suggest that NAB2 regulation represents a mechanism to guarantee transient EGR-1 activity following exposure of endothelial cells to VEGF and that NAB2 overexpression could be used to inhibit signals involved in the early phase of angiogenesis.

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“…Flow cytometric analysis of bladder tumor cells expressing GFP was carried out as previously described. 4 …”

Section: Flow Cytometric Analysismentioning

confidence: 99%

Visualizing superficial human bladder cancer cell growth in vivo by green fluorescent protein expression

et al. 2002

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There has been no reliable orthotopic model available to visualize the growth of human superficial bladder cancer over time and to evaluate the efficacy of intravesical therapies. We have developed a novel approach to accomplish this task by generating human superficial bladder tumor cells to stably express high levels of green fluorescent protein ( GFP ) in vivo. Superficial bladder tumors were produced in athymic mice by intravesical instillation. In our initial studies tumors were quantitated by image analysis at a single time point, and the results compared to the estimation of the percentage of GFP cells present using flow cytometry after obtaining single cell suspensions of normal and tumor cells in the same bladder. A high correlation between the two methods was seen. Therefore, in subsequent studies, approximately 1 week after the intravesical instillation of the GFP expressing cancer cells a small incision was made to expose the bladder. The anterior, posterior, and lateral images of each bladder were captured to visualize GFPexpressing tumors. The ratio of green fluorescence pixel area, which represented the tumor burden, to the total area of the bladder was then calculated. A similar procedure was performed at 2, 3, and 4 weeks after instillation of the tumor cells. Using this procedure tumor progression over time could be measured in each mouse. By using this approach, it will now be possible to monitor the initial tumor sizes in the bladder of each mouse and then to evaluate the efficacy of various intravesical therapy protocols including intravesical gene therapy alone or in combination with other treatment modalities. Cancer Gene Therapy ( 2002 ) 9, 681 -686 doi:10.1038/sj.cgt.7700489Keywords: bladder cancer; green fluorescent protein; image analysis B ladder cancer is the fifth most frequent cancer in the United States. More than 50,000 individuals are diagnosed with bladder cancer each year. Between 70% and 80% of these cases involve superficial tumor. Such lesions are usually managed with transurethral resection often followed by intravesical immunotherapy with Bacillus Calmette -Guerin ( BCG ). Although BCG can prolong the length of progression -free survival after the tumor is resected, approximately 50% of superficial lesions will continue to recur and as many as 30% will progress to a higher grade and /or stage. 1 Therefore, new therapeutic approaches, including gene therapy alone or in combination with other modalities of therapy, need to be considered for the treatment of refractory superficial bladder disease.We recently have developed an intravesical model to produce superficially growing human bladder tumors in athymic mice with high frequency. 2 In addition, our laboratory in collaboration with others has shown that this model can be used to document efficient intravesical adenoviral -mediated gene transfer when it is combined with the polyamide compound, Syn 3. 3 Our next challenge was to use this tumor model in vivo to examine the efficacy of various treatments and schedules. Howeve...

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Adenovirus-mediated expression of green fluorescent protein

Cited by 61 publications

References 9 publications

Inefficient killing of quiescent human epithelial cells by replicating adenoviruses: potential implications for their use as oncolytic agents

Inefficient killing of quiescent human epithelial cells by replicating adenoviruses: potential implications for their use as oncolytic agents

NAB2, a Corepressor of EGR-1, Inhibits Vascular Endothelial Growth Factor-mediated Gene Induction and Angiogenic Responses of Endothelial Cells

Visualizing superficial human bladder cancer cell growth in vivo by green fluorescent protein expression

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