Conditional expression of retrovirally delivered anti-MYCN shRNA as an in vitro model system to study neuronal differentiation in MYCN-amplified neuroblastoma

Using neuroblastoma cell lines to study neuronal differentiation in vitro is now well established. Several protocols, including exposure to various agents and growth factors, will differentiate neuroblastoma cell lines into neuron-like cells. These cells are characterized by a neuronal morphology with long extensively branched neurites and expression of several neurospecific markers.In this study we use retrovirally delivered inducible short-hairpin RNA (shRNA) modules to knock down MYCN expression in MYCN-amplified (MNA) neuroblastoma cell lines. By addition of the inducer doxycycline, we show that the Kelly and SK-N-BE(2) neuroblastoma cell lines efficiently differentiate into neuron-like cells with an extensive network of neurites. These cells are further characterized by increased expression of the neuronal differentiation markers NFL and GAP43. In addition, we show that induced expression of retrovirally delivered anti-MYCN shRNA inhibits cell proliferation by increasing the fraction of MNA neuroblastoma cells in the G1 phase of the cell cycle and that the clonogenic growth potential of these cells was also dramatically reduced.We have developed an efficient MYCN-knockdown in vitro model system to study neuronal differentiation in MNA neuroblastomas.Neuroblastoma is a childhood cancer arising from the sympaticoadrenal lineage of the neural crest. It is characterized by diverse clinical behaviours ranging from spontaneous regression, maturation to more benign forms (ganglioneuroblastoma or ganglioneuroma), to rapid tumour progression and death [1]. Amplification of the MYCN oncogene has been considered the most important prognostic factor for progressive disease and poor outcome. Despite intense efforts to elucidate a mechanism by which MYCN overexpression acts to promote the aggressive phenotype, the functional roles of the MYCN protein in neuroblastoma are poorly understood [2].Several alternative mechanisms for neuroblastoma regression have been proposed over