Neuroblastoma-specific cytotoxicity mediated by the Mash1-promoter and E. coli purine nucleoside phosphorylase.
BACKGROUND
Neuroblastoma is derived from cells of neural crest origin and often expresses the transcription factor human achaete-scute homolog 1 (HASH1). The aim of this study was to selectively kill neuroblastoma cells by expressing the suicide gene E. coli purine nucleoside phosphorylase (PNP) under the control of the Mash1 promoter, the murine homolog of HASH1.


METHODS
The E. coli PNP gene regulated by the Mash1 promoter was cloned into an expression vector and transfected into neuroblastoma and non-neuroblastoma cell lines. After addition of the prodrug M2-fluoroadenine 9-beta-D-arabinofuranoside (F-araA) the cell-specific toxicity was examined. To optimize the cell specific activity, different sizes of the Mash1 promoter were analyzed in neuroblastoma cell lines and compared with the activity in non-neuroblastoma cells.


RESULTS
Estimated as the percentages of CMV enhancer-promoter, the activity was significantly higher in the neuroblastoma cells, ranging from 17 to 58% when the shortest and the most active promoter was measured. The non-neuroblastoma cells yielded only 1-6% of the CMV promoter activity. When the shortest Mash1 promoter was combined with the E. coli PNP gene the cytotoxicity was 65% in the neuroblastoma cells with low cell death in the non-neuroblastoma cell lines, relative to the cytotoxicity where the E.coli PNP gene was regulated by the strong but non-specific CMV enhancer-promoter.


CONCLUSIONS
We show here that the Mash1 promoter regulating the PNP gene confers a cell-type selective toxicity in neuroblastoma cell lines. These results indicate the feasibility to use the Mash1 promoter for regulating E.coli PNP expression in gene-directed enzyme prodrug therapy (GDEPT) of neuroblastoma.
Arvidsson
Yvonne
Y
Department of Cell Biology, Institute of Anatomy and Cell Biology, Göteborg University, Gothenburg, Sweden. yvonne.arvidsson@llcr.med.gu.se
Sumantran
Venil
V
Watt
Fujiko
F
Uramoto
Hidetaka
H
Funa
Keiko
K
D051792:Basic Helix-Loop-Helix Proteins; D016923:Cell Death; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology; D015972:Gene Expression Regulation, Neoplastic; D018014:Gene Transfer Techniques; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D009447:Neuroblastoma / Q000235:genetics* / Q000473:pathology*; D011401:Promoter Regions, Genetic; D011683:Purine-Nucleoside Phosphorylase / Q000494:pharmacology*; D014157:Transcription Factors / Q000235:genetics* / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D051792:Basic Helix-Loop-Helix Proteins; D004268:DNA-Binding Proteins; D011683:Purine-Nucleoside Phosphorylase; D014157:Transcription Factors; C065500:ASCL1 protein, human