Expression of the reporter genes lacZ and EGFP in human glioblastoma cells using Listeria monocytogenes as vector for gene transfer.
OBJECTIVE
Glioblastoma has become a model target for viral gene therapy approaches. To circumvent some of the inherent problems of viral vectors, we examined the feasibility of Listeria monocytogenes for foreign gene expression in glioblastoma cells.


METHODS
The internalin-dependent uptake of L. monocytogenes into human glioma cell lines and into 22 cell cultures obtained during neurosurgery was studied by in vitro infection assays. Internalization rates of wild-type L. monocytogenes were compared with a deletion mutant and to four strains with plasmid-based recomplementation of the internalin A/B operon. For gene transfer experiments, plasmids encoding lacZ or EGFP under control of the cytomegalovirus promotor were transfected into Listeriae. Incubation times in cell culture were studied regarding infection of cells, intracellular listerial replication and reporter gene translation. Gene transfer efficiency was determined by beta-galactosidase activity and by fluorescence microscopy.


RESULTS
The internalin-defective strain (DeltainlAB2) showed less uptake compared with the wild type. An increased invasion rate was observed in strain DeltainlAB2 + inlB(PinlA) overexpressing internalin B; complementation of the entire operon (DeltainlAB2 + inlAB(PinlA)) revealed a synergistic effect. For gene transfer experiments into glioblastoma cells, the pathogenic strain 4bL312 was employed, and efficiencies up to 5% were achieved.


CONCLUSIONS
Internalins A and B are major determinants for listerial uptake into neuroepithelial tumors. The L. monocytogenes vector system enables foreign gene expression in glioblastoma cells. Ongoing research deals with optimization of transfection efficiencies, with the use of less pathogenic substrains and the cloning of suicide gene vectors.
Nestler
Ulf
U
Department of Neurosurgery, Justus-Liebig-University, Giessen, Germany. ulf.nestler@neuro.med.uni-giessen.de
Rollnik
Sebastian
S
Schulz
Chris
C
Chakraborty
Trinad
T
Domann
Eugen
E
Hain
Torsten
T
D001426:Bacterial Proteins / Q000235:genetics; D001932:Brain Neoplasms / Q000235:genetics; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D005909:Glioblastoma / Q000235:genetics* / Q000382:microbiology; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007763:Lac Operon; D008089:Listeria monocytogenes / Q000235:genetics* / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics; D009876:Operon / Q000235:genetics; D011401:Promoter Regions, Genetic; D017384:Sequence Deletion; D013997:Time Factors; D014764:Viral Proteins / Q000235:genetics
D001426:Bacterial Proteins; D008565:Membrane Proteins; D014764:Viral Proteins; C485184:enhanced green fluorescent protein; C095768:inlB protein, Listeria monocytogenes; C069761:internalin protein, Bacteria; D049452:Green Fluorescent Proteins