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. 2009 Jun;219(3):707-15.
doi: 10.1002/jcp.21719.

Adenovirus-mediated hPNPase(old-35) gene transfer as a therapeutic strategy for neuroblastoma

Tom Van Maerken  1 , Devanand Sarkar, Frank Speleman, Paul Dent, William A Weiss, Paul B Fisher
Affiliations

Adenovirus-mediated hPNPase(old-35) gene transfer as a therapeutic strategy for neuroblastoma

Tom Van Maerken et al. J Cell Physiol. 2009 Jun.

Abstract

Current treatment options for neuroblastoma fail to eradicate the disease in the majority of high-risk patients, clearly mandating development of innovative therapeutic strategies. Gene therapy represents a promising approach for reversing the neoplastic phenotype or driving tumor cells to self-destruction. We presently studied the effects of adenovirus-mediated gene transfer of human polynucleotide phosphorylase (hPNPase(old-35)), a 3',5'-exoribonuclease with growth-inhibitory properties, in neuroblastoma cells. Transgene expression was driven by either the cytomegalovirus (CMV) promoter or by a tumor-selective promoter derived from progression elevated gene-3 (PEG-3). Our data demonstrate that efficient adenoviral transduction of neuroblastoma cells and robust transgene expression are feasible objectives, that the PEG-3 promoter is capable of selectively targeting gene expression in the majority of neuroblastoma cells, and that hPNPase(old-35) induces profound growth suppression and apoptosis of malignant neuroblastoma cells, while exerting limited effects on normal neural crest-derived melanocytes. These findings support future applications of hPNPase(old-35) for targeted gene-based therapy of neuroblastoma and suggest that combination with the PEG-3 promoter holds promise for creating a potent and selective neuroblastoma therapeutic. J. Cell. Physiol. 219: 707-715, 2009. (c) 2009 Wiley-Liss, Inc.

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Figures

Fig. 1
Fig. 1
Infection with Ad.CMV-GFP induces GFP expression in neuroblastoma cells. A panel of five human neuroblastoma cell lines [IMR-32, NGP, SHEP, SK-N-BE(2c), and SK-N-SH], DU-145 human prostate carcinoma cells, and FM-516-SV immortalized human melanocytes were infected with Ad.CMV-GFP at the indicated MOIs and analyzed for the expression of GFP 24 h postinfection using flow cytometry. Columns represent the mean percentages of GFP-positive cells of three different experiments, and error bars indicate standard errors of the mean. Asterisk indicates percentage of GFP-positive cells 24 h after mock infection ranged from 0.19 to 0.22% for all cell lines.
Fig. 2
Fig. 2
Expression of hPNPaseold-35 in FM-516-SV human melanocytes and neuroblastoma cells following adenoviral gene transfer. FM-516-SV immortalized melanocytes and a panel of five neuroblastoma cell lines were infected with Ad.vec, Ad.CMV-hPNPase [Ad.CMV-hPNPaseold-35; hemagglutinin (HA)-tagged hPNPaseold-35 expression driven by the CMV promoter], or Ad.PEG-hPNPase (Ad.PEG-hPNPaseold-35; HA-tagged hPNPaseold-35 expression driven by the PEG-3 promoter) at the indicated MOIs, and probed 48 h postinfection for hPNPaseold-35 expression by Western blot analysis using an anti-HA antibody. Expression of EF1α is shown as loading control.
Fig. 3
Fig. 3
Effect of Ad.vec, Ad.CMV-hPNPaseold-35, and Ad.PEG-hPNPaseold-35 on growth and viability of FM-516-SV human melanocytes and neuroblastoma cells. Exponentially growing cells were untreated or infected with Ad.vec, Ad.CMV-hPNPase (Ad.CMV-hPNPaseold-35), or Ad.PEG-hPNPase (Ad.PEG-hPNPaseold-35) at 100 pfus/cell, and the number of viable cells was determined at 1, 3, and 5 days postinfection using MTT assay. Columnsrepresentthemeanrelativenumbersofviablecellsofthreeindependentexperiments, and error bars indicate standard errors of the mean.
Fig. 4
Fig. 4
Effect of Ad.vec, Ad.CMV-hPNPaseold-35, and Ad.PEG-hPNPaseold-35 on cell cycle distribution and hypodiploid DNA content of human neuroblastoma cells. NGP (A), SK-N-SH (B), and SHEP (C) cells were untreated or infected with Ad.vec, Ad.CMV-hPNPase (Ad.CMV-hPNPaseold-35), or Ad.PEG-hPNPase (Ad.PEG-hPNPaseold-35) at 100 pfus/cell, harvested at 2, 3, 4, and 5 days postinfection and monitored for DNA content by propidiumiodide staining and flow cytometric analysis. Columns represent the mean cell cycle and apoptotic fractions of three different experiments, and error bars correspond to standard errors of the mean.
Fig. 5
Fig. 5
Effect of Ad.vec, Ad.CMV-hPNPaseold-35, and Ad.PEG-hPNPaseold-35 on caspase-3 and caspase-7 activity in human neuroblastoma cells. NGP, SK-N-SH, and SHEP cells were untreated or infected with Ad.vec, Ad.CMV-hPNPase (Ad.CMV-hPNPaseold-35), or Ad.PEG-hPNPase (Ad.PEG-hPNPaseold-35) at 100 pfus/cell, and the combined activity of caspase-3 and caspase-7, relative to uninfected cells, was determined at 2 and 3 days postinfection. Columns represent the mean caspase-3 and caspase-7 activity values of triplicate wells, and error bars correspond to standard deviations.
Fig. 6
Fig. 6
Adenoviral infection induces neuronal differentiation of NGP human neuroblastoma cells. A: Phase-contrast images of NGP cells left untreated for 5 days (left) and 5 days after infection with Ad.vec (right). Adenoviral infection induced clear morphological signs of neuronal differentiation, including a polar morphology and extensive outgrowth of neuritic processes, which formed a net-like arrangement. Similar morphological changes were observed after infection of NGP cells with Ad.CMV-hPNPaseold-35 or Ad.PEG-hPNPaseold-35 (not shown). B: Real-time quantitative RT-PCR analysis of expression of neuronal differentiation markers (NEFH, NEFL, NEFM, and CHAT) in NGP cells harvested 5 days after exposure to control conditions or to infection with Ad.vec, Ad.CMV-hPNPase (Ad.CMV-hPNPaseold-35), or Ad.PEG-hPNPase (Ad.PEG-hPNPaseold-35) at 100 pfus/cell. Columns represent the mean mRNA expression levels derived from two RT-PCR measurements, and error bars indicate standard deviations. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
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