Abstract
Neuroblastomas are the second most common solid tumor in children but the molecular mechanisms underlying the initiation and progression of this disease are poorly understood. We previously showed that the Brn-3b transcription factor is highly expressed in actively proliferating neuroblastoma cells but is significantly decreased when these cells are induced to differentiate. In this study, we analyzed the effects of manipulating Brn-3b levels in the human neuroblastoma cell line, IMR-32 and showed that constitutive overexpression of Brn-3b consistently increased cellular growth and proliferation in monolayer as well as in an anchorage-independent manner compared with controls whereas stably decreasing Brn-3b can reduce the rate of growth of these cells. Cells with high Brn-3b also fail to respond to growth inhibitory retinoic acid, as they continue to proliferate. Moreover, Brn-3b levels significantly modified tumor growth in vivo with elevated Brn-3b resulting in faster tumor growth in xenograft models whereas decreasing Brn-3b resulted in slower growth compared with controls. Interestingly, elevated Brn-3b levels also enhances the invasive capacity of these neuroblastoma cells with significantly larger numbers of migrating cells observed in overexpressing clones compared with controls. Because invasion and metastasis influence morbidity and mortality in neuroblastoma and so significantly affect the course and outcome of neuroblastomas, this finding is very important. Our results therefore suggest that Brn-3b transcription factor contributes to proliferation of neuroblastoma cells in vivo and in vitro but may also influence progression and/or invasion during tumorigenesis. It is possible that decreasing Brn-3b levels may reverse some effects on growth and proliferation of these cells.
Highlights
Cells with Different Levels of Brn-3b—Because Brn-3b transcription factor appears to be associated with proliferation of neuroblastoma cells, we wished to study the effect of manipulating its expression in these cells as this approach provides a powerful tool to analyze its regulatory role in these cells
Neuroblastoma tumors arise from neural crest cells destined for sympathetic neuronal lineage or the adrenal medulla [2, 29]
Because of the remarkable clinical heterogeneity of neuroblastomas, it is important to identify factors that might be involved in initiation and development of this complex disease as this could increase our understanding of the factors that underlie the diversity observed as well as provide possible markers which may be useful for diagnosis and/or prognosis, or could be targeted for more effective therapy
Summary
The eukaryotic expression vector pLTR, contained the MoMuLV promoter driving expression of either cDNA encoding either Brn-3b(s) (Brn-3b expression vector) [18], or 300 bases of Brn-3b cDNA in the antisense orientation (common to both Brn-3b(s) and Brn-3b(l) (Brn-3b antisense)) [18]. pCI neo plasmid was obtained from PromegaTM Ltd. The eukaryotic expression vector pLTR, contained the MoMuLV promoter driving expression of either cDNA encoding either Brn-3b(s) (Brn-3b expression vector) [18], or 300 bases of Brn-3b cDNA in the antisense orientation (common to both Brn-3b(s) and Brn-3b(l) (Brn-3b antisense)) [18]. PCI neo plasmid was obtained from PromegaTM Ltd. 2 S.
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