[Oncogen N-myc expression and measurement of DNA ploidy in neuroblastoma: a double staining flow cytometric analysis]

When N-myc copy number was assessed by molecular biology, it has been proven that an amplification of the oncogene was a bad prognosis in childhood neuroblastoma, and the same goes for DNA-diploidy. This study concerns the development of a biparametric flow cytometric analysis of 2 neuroblastoma cell lines (SK-N-SH and IGR-N-91), which exhibited respectively 1 and 60 copies of the N-myc oncogene. An indirect immunofluorescence technique allowed N-myc oncoprotein staining and an isotypic control was used to assess the threshold of specific fluorescence. Simultaneously, a double staining with propidium iodide gave the nuclear DNA content. For both types of cells, the level of N-myc expression was calculated as a fluorescence index (IF). IF for IGR-N-91 appeared 2.5 times higher than those of SK-N-SH. This fluorescence index increases significantly during the exponential growth of N-myc amplified cells, whereas it does not vary for SK-N-SH. During IGR-N-91 tumoral evolution, the cell line which derived from murine heart metastasis was the only one to show an increased IF. When applied to 10 neuroblastoma cell suspensions, this double staining showed an high IF for only 1 N-myc amplified case. A poor cell yield after tumoral dissociation and too much debris did not allowed the calculation of IF for half of them, which hampered a routine development of this technique.