Evaluation of LINE-1 mobility in neuroblastoma cells by in vitro retrotransposition reporter assay: FACS analysis can detect only the tip of the iceberg of the inserted L1 elements.
Long Interspersed Nuclear Elements (L1) are retroelements generally repressed in most differentiated somatic cells. Their activity has been observed in some undifferentiated and tumour cells and could be involved in tumour onset and progression. Growing evidences show that the L1 activation can occur in neuronal precursor cells during differentiation process. Neuroblastoma is a tumour originating from neuronal precursor cells, and, although the molecular basis of its progression is still poorly understood, the implication of L1 activation has not yet been investigated. In this study L1 mobility in neuroblastoma BE(2)C cells was assessed using the in vitro retrotransposition assay consisting in an episomal EGFP-tagged L1(RP) element, whose mobility can be evaluated by cytofluorimetric analysis (FACS) of EGFP expression. FACS results have shown a low retrotransposition activity. To detect L1(RP) integrated in transcriptionally repressed genomic sites, both a cell treatment with a stimulator of reporter gene promoter, and a quantitative Real-Time PCR analysis were performed. A retrotransposition activity ten and one thousand times that of FACS was found, respectively. These results point out that the real rate of L1 retrotransposition events in tumour cells might be considerably higher than that reported so far by evaluating only the reporter gene expression.
Del Re
Brunella
B
Department of Evolutionary Experimental Biology, University of Bologna, Bologna, Italy. brunella.delre@unibo.it
Marcantonio
Pamela
P
Capri
Miriam
M
Giorgi
Gianfranco
G
D045744:Cell Line, Tumor; D003587:Cytomegalovirus / Q000235:genetics; D005434:Flow Cytometry; D018628:Gene Dosage; D017930:Genes, Reporter / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D020084:Long Interspersed Nucleotide Elements / Q000235:genetics*; D009447:Neuroblastoma / Q000235:genetics* / Q000473:pathology; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*; D020218:Response Elements / Q000187:drug effects / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D014212:Tretinoin