Introduction of in vitro transcribed ENO1 mRNA into neuroblastoma cells induces cell death.
BACKGROUND
Neuroblastoma is a solid tumour of childhood often with an unfavourable outcome. One common genetic feature in aggressive tumours is 1p-deletion. The alpha-enolase (ENO1) gene is located in chromosome region 1p36.2, within the common region of deletion in neuroblastoma. One alternative translated product of the ENO1 gene, known as MBP-1, acts as a negative regulator of the c-myc oncogene, making the ENO1 gene a candidate as a tumour suppressor gene.


METHODS
Methods used in this study are transfection of cDNA-vectors and in vitro transcribed mRNA, cell growth assay, TUNEL-assay, real-time RT-PCR (TaqMan) for expression studies, genomic sequencing and DHPLC for mutation detection.


RESULTS
Here we demonstrate that transfection of ENO1 cDNA into 1p-deleted neuroblastoma cell lines causes' reduced number of viable cells over time compared to a negative control and that it induces apoptosis. Interestingly, a similar but much stronger dose-dependent reduction of cell growth was observed by transfection of in vitro transcribed ENO1 mRNA into neuroblastoma cells. These effects could also be shown in non-neuroblastoma cells (293-cells), indicating ENO1 to have general tumour suppressor activity. Expression of ENO1 is detectable in primary neuroblastomas of all different stages and no difference in the level of expression can be detected between 1p-deleted and 1p-intact tumour samples. Although small numbers (11 primary neuroblastomas), there is some evidence that Stage 4 tumours has a lower level of ENO1-mRNA than Stage 2 tumours (p = 0.01). However, mutation screening of 44 primary neuroblastomas of all different stages, failed to detect any mutations.


CONCLUSIONS
Our studies indicate that ENO1 has tumour suppressor activity and that high level of ENO1 expression has growth inhibitory effects.
Ejeskär
Katarina
K
Dept, Clinical Genetics, University of Gothenburg, Sahlgrenska University Hospital/East, SE-416 85 Gothenburg, Sweden. katarina.ejeskar@clingen.gu.se
Krona
Cecilia
C
Carén
Helena
H
Zaibak
Faten
F
Li
Lingli
L
Martinsson
Tommy
T
Ioannou
Panayiotis A
PA
D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000502:physiology; D014408:Biomarkers, Tumor / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D017353:Gene Deletion; D016147:Genes, Tumor Suppressor; D005822:Genetic Vectors; D016678:Genome; D006801:Humans; D020287:In Situ Nick-End Labeling; D066298:In Vitro Techniques; D008957:Models, Genetic; D009154:Mutation; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism*; D010751:Phosphopyruvate Hydratase / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; D014408:Biomarkers, Tumor; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; C095157:ENO1 protein, human; D010751:Phosphopyruvate Hydratase